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被誉为“灰度之子”的LPT接连翻倍、到底还有多少空间？ - 知乎

被誉为“灰度之子”的LPT接连翻倍、到底还有多少空间？ - 知乎切换模式写文章登录/注册被誉为“灰度之子”的LPT接连翻倍、到底还有多少空间？阿佳之前一直推荐的潜力币LPT又涨了个翻倍，无疑是今天币界最靓的仔，目前一直持续上涨趋势，LPT 网络价格最高上涨117.14%，截止发文，稳定在85.39%的涨幅。在过去不到一个月时间里，从13U位置左右一直陆续暴涨，截止目前最高位置已经飙升至71U左右,真是把牛市表现体现的淋漓尽致，LPT不是每天都给大家惊喜，所以路上很多耐不住寂寞的追随者半路下车，今天看到涨幅应该也会拍断大腿咯，我不说大话，这个币我是看到冲上100的，好了，话不多说，接下来就带你了解这个LPT。LPT——“灰度之子”其实说到LPT，不得不提它的另一个名字，“灰度之子”。灰度是一个机构，作为币圈的风向标，旗下有13枝信托基金，其中lpt的市值是最小的。我们都知道灰度投资的币种有很多，但是能上灰度信托清单的也就这十三个，而lpt又是当前市值最小的，能被灰度选中加入投资组合，可见意义深远。要知道上了灰度信托基金的币种最少都有10倍的涨幅，lpt灰度持仓不在少数，不可能默默无闻的。而且灰度曾经还有增仓的动作，所以它的热度一直都是中等偏上的，我们从持币地址数可以看出来，有了灰度基金加持之后，它的地址数量飙升至百万级别了，截至目前达到了2530750个。一般来讲，持币地址数不过万不能玩，那么这个百万级别的地址数肯定是可以玩一玩的。Livepeer Token (LPT) 是什么首先来看看Livepeer 的基本信息。它是做基于以太坊的开源视频基础设施服务的，主要面向视频工程师提供去中心化的编码服务。它的原理是把编码的任务分发给网络上的节点，节点通过抵押代币并完成编码任务获得奖励，进而对终端视频用户提供服务。公认的优点 :1. 视频编码需要的计算量大，适合分布式网络应用2. 获得多家知名 VC 投资（这点是最重要的，也是它最大的优势）3. 在以太坊生态中较为活跃缺点 :1. 与竞品相比经济模型及技术均缺乏亮点，代币价值来源有限2. 视频解码仅仅是视频应用的一小部分，没有提供完整生态服务（也许可以当在优点，因为专注而专业）3. 团队缺乏产品及运营背景4。整个团队也过于偏技术，只有一名产品经理，比较难设计出优秀的产品，也没有较强的运营能力。5 LPT 代币并不用于广播者向网络发送任务，广播者使用 ETH 来支付费用。这一点大大降低了 LPT 代币的使用场景，使其仅限于网络的 staking。Livepeer 的经济模型：广播者-发布原创视频流编码者-将视频流编码成不同的制式、清晰度和格式传递者-分发视频和协议相关的信息消费者-视频流的需求方，观看或者传递给 dapp 用户抵押者-将代币抵押给网络并投票给编码者，可以同时是编码者Livepeer的价值所在：工程在开发过程中，开发者可以添加直播的和随选的视频；内容创造者可以看的直播内容多元丰富，包括视频、游戏、编码、娱乐以及教育性的课程； Livepeer的节点都可以创造直播内容，又通过转码视频格式可以赚得Livepeer Token (LPT)；直播者可以进行质管，同时控制基础结构的费用。Livepeer 的投资机构根据 Crunchbase 的信息， 2017 年 5 月 Livepeer 获得种子轮融资，参投方包括 Distributed Global、Digital Currency Group、Charge Ventures 等机构，投资数额不详。这些机构不要说你们不认识，随便一个放点份额出来市场都要疯狂。更让人无法理解的是2019 年 6 月 Livepeer 的 A 轮融资中，又有 8 家机构参投，其中包括 Digital Currency Group、Compound、Multicoin 等知名币圈机构，投资数额共 800 万美元。其融资阵容可以说相当豪华，并且在代币价格表现不佳的情况下获得新一轮融，这下理解我之前说自己实际在车上的理由吧。项目愿景：Livepeer项目旨在提供一种完全去中心化、高度可扩展、加密Token激励的实时视频流网络协议，并产生一种解决方案，该解决方案可以作为去中心化式开发（Web3）堆栈中的实况媒体层。此外，LIVEPER旨在为任何现有的直播提供一种经济高效的集中直播解决方案。随着LIVER对等技术和协议的交付，它将使用户能够参与以下流程：在相机、电话、屏幕或网络摄像头上捕获视频并将其发送到Livepeer网络。在网络中运行的节点将把它编码成所有必需的格式，以达到每一个支持的设备。运行这些节点的用户将通过ETH广播支付的费用来激励，并有机会通过协议Token来建立声誉，以赚取将来执行更多工作的权利。网络上的任何用户都可以请求查看流，并且它将在近实时地自动分发给它们。最后我想说Livepeer 作为区块链视频领域的项目，希望发展成为以太坊平台上的去中心化视频基础设施服务 . 该项目在区块链视频赛道上，要超越的对手还不少。但灰度后面连续三个持仓跟当前币种联系起来，真的有无尽的想像。总的来说，Livepeer在背景方面显示出较强的实力，我们拭目以待。好了，今天就分享到这里吧！最后！我们主要重点提供海外一级市场优质项目和二级市场优质暴涨币，最新ido消息分享，不收费，目的为大家提供一个高质量的前沿资讯社区我们一直在努力对接更多币圈一线机构与项目方，崛起社区的理念就是双赢，后续会发车更多海内外一级市场优质项目，私信我期待你的加入！发布于 2021-11-09 20:37比特币 (Bitcoin)LTP区块链(Blockchain)赞同 1添加评论分享喜欢收藏申请

安装打印机时LPT，COM、USB端口区别_lpt端口和usb端口区别-CSDN博客

安装打印机时LPT，COM、USB端口区别

最新推荐文章于 2023-12-28 14:50:07 发布

wudashuang

最新推荐文章于 2023-12-28 14:50:07 发布

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本文链接：https://blog.csdn.net/wudashuang/article/details/100881206

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LPT(并口):LPT是用于使用打印机或其它设备的。LPT并口是一种增强了的双向并行传输接口，在USB接口出现以前是扫描仪，打印机最常用的接口。(目前使用滴少) COM（串口）:COM端口通常用于连接鼠标及通讯设备（如连接外置式MODEM进行数据通讯）等(不用安装驱动) 并行接口和串行接口区别并行口与串行口的区别是交换信息的方式不同，并行口能同时通过8条数据线传输信息，一次传输一个字节；而串行口只能用1条线传输一位数据，每次传输一个字节的一位。并行口由于同时传输更多的信息，速度明显高于串行口，但串行口可以用于比并行口更远距离的数据传输。

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安装打印机时LPT，COM、USB端口区别

LPT(并口):LPT是用于使用打印机或其它设备的。LPT并口是一种增强了的双向并行传输接口，在USB接口出现以前是扫描仪，打印机最常用的接口。(目前使用滴少)COM（串口）:COM端口通常用于连接鼠标及通讯设备（如连接外置式MODEM进行数据通讯）等(不用安装驱动)并行接口和串行接口区别并行口与串行口的区别是交换信息的方式不同，并行口能同时通过8条数据线传输信息，一次传输一个字节；而串行口...

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串口及硬件信息查询（com和lpt）

05-21

能够查找出计算机所有端口设备，并同时提供设备对应的硬件ID信息，能够轻松判定设备的拔插、寻找、绑定等

LPT接口打印测试

11-24

LPT接口打印测试，只能支持LPT接口的打印测试。可以配合我的另一个资源“LPT接口共享USB命令”使用，达到测试USB接口的功能。

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LTPI协议的理解——1、LTPI协议的定义和结构

gzy0506的博客

12-28

1433

定义DC-SCM 2.0 LTPI 结构GPIO通道I2C/SMBus通道Uart通道OEM通道数据通道总结LTPI (LVDS Tunneling Protocol & Interface，LVDS 隧道协议和接口)是一种协议和接口，设计用于在 HPM 和 SCM 之间传输各种低速信号。它不仅支持 GPIO 的隧道传输，还支持 SMBus、I2C、UART 等低速串行接口的隧道传输。它还可以通过额外的专有 OEM 接口进行扩展，并为 HPM CPLD 和 SCM CPLD 之间的原始数据隧道提供支持。

通过USB、LTP端口调用Zebra打印

01-02

通过USB、LTP端口调用Zebra打印，斑马打印助手，支持LPT/COM/DRV三种模式，适用于标签、票据、条码打印。

portlist:端口列表-列出Microsoft Windows 2000或更高版本（XP，Vista，7、8或10）上可用的（可选的，记住的所有）COM（串行或调制解调器）或LPT（打印机）端口

05-09

端口列表

端口列表-列出Microsoft Windows 2000或更高版本（XP，Vista，7、8或10）上可用的（可选的，记住的所有）COM（串行或调制解调器）或LPT（打印机）端口。

端口列表

目的

将新的USB或蓝牙（等）串行或并行设备连接到运行Microsoft Windows的PC的一个问题是，您并不总是看到闪烁的消息，该消息告诉您串行或并行端口的名称。您可能会忘记名称，或者如果将其插入其他USB端口，则名称可能会更改，或者使用该端口的程序在重新连接设备之前没有关闭该端口。

这也会影响假装成USB串行或并行端口的事物。 GPS接收器或Arduino等微控制器开发设备。

除其他功能外，报告的列表还可以限于选定的USB VID（供应商ID）或VID：PID对（供应商和产品ID）。长（-l）和详细（-v）模式报告的信息超出了简单的端口列表，有助于调试驱动程序.inf

并口打印口lpt编程

03-07

一定有你有用的内容，

“(编程控制示例）

// 此段代码为并口向打印机进行写数据，并发送控制信息。

#define LPT_CLEAR_MASK 0x40

#define LPT_STROBE_HI 0x0D

#define LPT_STROBE_LO 0x0C

#define LPT_STATUS_BITS 0xF8

#define LPT_BITS_INVERT 0x48

#define LPT_NOTBUSY 0x80

#define LPT_PAPEROUT 0x20

#define LPT_SELECT 0x10

#define LPT_NFAULT 0x08

”

Yolov4部署到ZYNQ系列1-USB转UART驱动不在COM和LPT显示问题的解决方案

weixin_41029027的博客

09-04

1550

USB转UART驱动不在COM和LPT显示问题的解决方案，Zynq Ultrascale+ MPSoC连接UART转USB接口到win10笔记本时出现的问题。

LPT,COM口

weixin_30443747的博客

11-24

552

打印机有两种接口,一种是COM接口,还有一种是USB接口.USB接口通常是用在喷墨打印机上的,而COM接口是

用在针字打印机和激光印字机,不过激光印字机也有USB接口。

COM端口是多功能的端口，还可以接游戏手柄、鼠标（老式的）等其它设备。

LPT接口和com口的不同LPT端口为打印机专用端口，lpt并口是一种增强的双向并行传输接口。

转载于:https://www.cnblogs.com/...

win10 企业版设备管理器找不到端口(COM和LPT)

LPT - What does LPT stand for? The Free Dictionary

LPT - What does LPT stand for? The Free Dictionary https://acronyms.thefreedictionary.com/LPTPrinter Friendly

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LPT Also found in: Encyclopedia, Wikipedia.

Category filter: Show All (52)Most Common (1)Technology (11)Government & Military (7)Science & Medicine (17)Business (11)Organizations (11)Slang / Jargon (2)AcronymDefinitionLPTLocal Printer TerminalLPTLife Pro TipsLPTLanguage Proficiency Test (various locations)LPTLine PrinterLPTLicensed Professional TeacherLPTLow Pressure TurboLPTLower Precision TargetsLPTLow Pressure TurbineLPTLicensed Physical TherapistLPTLicensed Penetration TesterLPTLight Pressure Turbo (automotive)LPTLaw Practice TodayLPTLinear Position Transducer (medical equipment)LPTLower Paxton Township (Pennsylvania)LPTListed Property TrustLPTLicensed Psychiatric TechnicianLPTLondon Probation Trust (UK)LPTLongest Processing Time (operations management)LPTLorain Palace Theater (Lorain, OH)LPTLocal Positioning TechnologyLPTLoss Portfolio Transfer (financial reinsurance)LPTLine Printer TerminalLPTLow Power TransceiverLPTLow Profile Turret (gun)LPTLoss Prevention TeamLPTLogistics Preparation of the TheaterLPTLiquid Penetrant Test (non destructive testing)LPTLimited Phlebotomy Technician (licensed medical assistant)LPTLow Platform TrailerLPTLinear Planarization Technology (semiconductors)LPTLower-Order Path TerminationLPTLimited Performance TestLPTLovelock Paiute Tribe (Nevada)LPTLunar Propellant TankerLPTLabor Process TheoryLPTLoad Port TransferLPTLamina Propria T CellLPTLow Pressure TestLPTLimited Production TestLPTLaser Propulsion TestLPTLimited Private TypeLPTLandings Per Tire (aviation)LPTLouisville's Play Toy (Matt Hayes)LPTLegal Professional TrainingLPTLow Probability TargetingLPTLocal Platform Testing (Sprint)LPTLogistic Plug TrunkLPTLow Power TechniqueLPTLow Profile TransformerLPTLimited Power Technician (Minnesota State Board of Electricity license)LPTLiterary Present Tense (language use)LPTLength-Preserving Transform (lossless text transformation)Copyright 1988-2018 AcronymFinder.com, All rights reserved.

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To register an interest in attending, please email Gemma.Wooster@leicspart.nhs.uk You can hear more about working in therapy at LPT by watching this video at https://youtu.be/ tfOd5LJCSTQ To browse all our vacancies, visit http://bit.Physio staff open day at hospital; IN BRIEFThe LPT business unit of Lanxess currently employs over 500 people worldwide.Lanxess to increase prices for some productsThe interplay between the time value discount and the LPT risk load can be seen where these structures have the same point of exhaustion.Insurers Increasingly Seek Legacy Transfers to Shed Long-Tail LiabilitiesAccess Contracting will be integrated within PTSG's Access and Safety Division and LPT into PTSG's Electrical Services Division.PTSG Acquires UK-based Lightning Protection Testing Firm in Cash DealAt assessment, LPT children were 36 kg and 138 cm tall on average, with a head circumference of 54 cm.Late preterm births tied to neurodevelopmental issuesLuminescent Paint Technologies (LPT) is an invention-oriented technical company.New differentiated products might be disruptive as well: a discussion with the developers of two game-changing technologies"We reiterate our original assertion that the failure of LPT is in no way down to the people concerned, but to external financial pressures affecting shippers and customers alike which affected base costs and margins right through the supply chain."Jobs setback as port produce terminal axed a second timeIn fact, our maintenance instructions require us to have both mainmounts chocked and three tie-down chains installed prior to doing an LPT.Runaway prowlerCRISIL has revised its rating outlook on the long-term bank facilities of Luminous Power Technologies Pvt Ltd (LPT, part of the Luminous group) to 'Positive' from 'Stable', while reaffirming the rating at 'A+'; the rating on the short-term facility has been reaffirmed at 'P1'-CRISIL changes Luminous Power Technologies outlook to "positive"Soni further informed that under this proposed scheme, three HPTs are to be set up at Sirpath, Jasrota Top and Khalsar in Kashmir, while one LPT for FM radio programmes would come up at Naushera.New broadcasting and relay transmitters to come up in remote areas of J-KIn a conventional engine configuration, the fan, the low-pressure compressor (LPC) and the low-pressure turbine (LPT) are all mechanically attached by a shaft, so turning at the same speed.Budget flight plan: Pratt and Whitney has develop an aeroengine that promises to deliver a much-needed cut in fuel cost

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LPT是什么意思? - LPT的全称 | 在线英文缩略词查询

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首页 › 3 个字母 › LPT

LPT 是什么意思?

你在寻找LPT的含义吗？在下图中，您可以看到LPT的主要定义。如果需要，您还可以下载要打印的图像文件，或者您可以通过Facebook，Twitter，Pinterest，Google等与您的朋友分享。要查看LPT的所有含义，请向下滚动。完整的定义列表按字母顺序显示在下表中。

LPT的主要含义

下图显示了LPT最常用的含义。您可以将图像文件下载为PNG格式以供离线使用，或通过电子邮件发送给您的朋友。如果您是非商业网站的网站管理员，请随时在您的网站上发布LPT定义的图像。

LPT的所有定义

如上所述，您将在下表中看到LPT的所有含义。请注意，所有定义都按字母顺序列出。您可以单击右侧的链接以查看每个定义的详细信息，包括英语和您当地语言的定义。

首字母缩写词定义LPT上市的地产信托LPT今天的法律实践LPT低功率的收发信机LPT低功耗技术LPT低压涡轮LPT低帕克斯顿乡镇LPT低平台挂车LPT低概率目标LPT低气压试验LPT低调变压器LPT低阶通道终结LPT保持长度不变的变换LPT光压涡轮LPT农历的推进剂油轮LPT劳动过程理论LPT持牌的渗透测试仪LPT持牌的物理治疗师LPT持牌的精神科技术员LPT损失的投资组合转移LPT损失预防小组LPT文学现在时态LPT最长的处理时间LPT有限的专用类型LPT有限的性能测试LPT有限的放血技术员LPT有限的生产测试LPT本地平台测试LPT本地打印机终端LPT权力有限的技术员LPT每胎降落LPT法律专业培训LPT洛雷恩宫殿剧院LPT液体渗透检测LPT激光推进试验LPT物流插头树干LPT物流的剧院的制备LPT粘膜固有层 T 细胞LPT行式打印机LPT行式打印机终端LPT装货港转移LPT路易斯维尔的玩具

‹ LPX

LQ ›

语言

简体中文

Licensed Penetration Tester (Master) | LPT (Master) | CPENT | EC-Council

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CERTIFIED PROFESSIONALSIN 145 COUNTRIES

CERTIFIED PROFESSIONALS

IN 150 COUNTRIES

Become A Licensed Penetration Tester Master (LPT)

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Introducing The World’s Most Advanced

Penetration Testing Program

The Certified Penetration Testing Professional (C|PENT) program by EC-Council was created to prepare those that want to be recognized as elite penetration testing professionals. Our training has been designed by the best in the industry and is meant to push you to develop the kind of skill that you’ve been waiting to acquire.L|PT (Master) training (via (C|PENT)) is not comfortable (and the exam is even worse!), but filled with intense stress meant to elicit the best from you. Those who prevail will have developed an instinctual response to real-world penetration testing challenges..

Become L|PT Master

The L|PT (Master) Certification Program

This exam has one purpose:

To differentiate the experts from the novices in Penetration Testing!

There are good penetration testers and then there are great penetration testers.

Unless you are bent on being nothing other than the best in penetration testing, don’t bother registering for this program as you are probably not cut out for it.

This is the first program in the world that can give you two potential outcomes in one single exam!

If you are one of the very few top-notch experts in the game that can test some of the most hardened systems in the world, then you may have what it takes to become a Licensed Penetration Tester Master. To earn the L|PT (Master) you will need to score at least 90% on our 24-hour exam.

Even if you don’t score 90% on the exam, which is a rare feat, candidates that score more than 70% will earn the C|PENT certification.

And you must know that while you are racing against time, you will be under the watchful eyes of the EC-Council proctors who will be online and live! This added pressure will test your mental strength.

Your pen-testing skills will be challenged against a multi-layered network architecture with defense-in-depth controls. You will be required to make decisions under immense pressure at critical stages while selecting your approach and exploits.

As you progress, you will need to maneuver web applications and host penetration testing tools and tricks in an internal and external context to ultimately pwn the hosts and exfiltrate data required to complete the challenges.

The exam will require you to demonstrate mastery of deploying advanced pen-testing techniques and tools including multi-level pivoting, OS vulnerabilities exploits, SSH tunneling, host-based application exploits, privilege escalation, and web server and web application exploitation such as arbitrary local and remote file upload, SQL injection, and parameter manipulation, etc. – all in a real-life scenario on hardened machines, networks, and applications.

You will be facing the ticking clock and there’s no time to hesitate. There’s no time for second-guessing. Try either of these and be prepared to fail!

Program Information

What's New in L|PT Master

Course Outline

Who is it for?

Brochure

What's New in L|PT Master

What’s New in L|PT Master

There are good penetration testers and then there are great penetration testers.

Unless you are bent on being nothing other than the best in penetration testing, don’t bother registering for this program as you are probably not cut out for it.

This is the first program in the world that can give you two potential outcomes in one single exam!

In this training program, you will learn professional security and penetration testing skills. The course is designed to show advanced concepts like advanced windows attacks, attacking IoT systems, and writing exploits with advanced binary exploitation to go beyond flat networks. You will learn to bypass a filtered network, pentest OT systems, access hidden networks with pivoting, double pivot, escalate privilege, and evade defense mechanisms.

The Planet’s Most Advanced Penetration Testing Range

This advanced penetration testing exam from EC-Council is built on the backbone of the Advanced Penetration Testing Cyber Range (ECCAPT) and was designed by experts who each have more than 25 years of professional security testing across the globe.

You will be exposed to various advanced penetration testing concepts such as fuzzing, PowerShell scripting, BASH, Python, Perl, and Ruby environments, scripting, and mobile device penetration testing, among others.

Course Outline

Module 01: Demonstrate a repeatable and measurable approach to penetration

testing

Module 02:Perform advanced techniques and attacks to identify SQL injection,

Cross-site scripting (XSS), LFI, RFI vulnerabilities in web applications

Module 03: Submit a professional and industry-accepted report that achieves

management and technical buy-in

Module 04: Get access to proprietary EC-Council penetration testing

methodologies

Module 05: Write exploit codes to gain access to a vulnerable system or

application

Module 06: Exploit vulnerabilities

in Operating systems such as Windows,

Linux

Module 07: Perform privilege escalation to gain root access to a system

Module 08: Demonstrate ‘Out-of-the-box’ and ‘lateral’ thinking

Module 09: Ensure the integrity and value of

the penetration testing certification,

in a fully online, remotely proctored certification

exam

Who is it for?

Who Is It For?

Ethical Hackers

Penetration Testers

Network Server Administrators

Firewall Administrators

Security Testers

System Administrators and Risk Assessment Professionals

Cybersecurity Forensic Analyst

Cyberthreat Analyst

Cloud Security

Analyst Information Security Consultant

Application Security Analyst

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Brochure

Making an informed decision is difficult, and that’s where EC-Council’s LPT brochure comes to your rescue. The Licensed Penetration Tester (Master) credential is the most trusted penetration testing certification that employers worldwide value, and for good reasons.The comprehensive curriculum covers advanced Windows attacks, attacking IoT systems, writing exploits with advanced binary exploitation, pentesting OT systems, and many more advanced concepts.When you successfully achieve the LPT (Master) certification, you will gain professional security and penetration testing skills.This is only an overview of LPT (Master) and what you will learnFor complete information, download the brochure now.Download Brochure

Is This Course For Me?

How do you create an Advanced Penetration Tester?

IoT Penetration Testing

This is the first certification that requires you to locate IoT device(s) then gain access to the network. Once on the network, you’ll be challenged to identify the firmware of the IoT device, extract it, and then reverse engineer it by extracting the file system, analyzing the content and the files, mounting the file system, and booting it with Firmadyne.

OT and SCADA Penetration Testing

The test contains a zone dedicated to ICS/SCADA networks that you will have to penetrate from the IT network side and gain access to the OT network. Once there, you will have to identify the Programmable Logic Controller (PLC) and then modify the data to impact the OT network. To do this you must be able to intercept the Mod Bus Communication protocol and communication between the PLC and other nodes. If you can’t do that presently, then you should enroll in the C|PENT program to learn!

Binary Analysis and Exploitation

Unlike other certifications, this will not just be a simple 32-bit code, but there will be 32- AND 64-bit code challenges and some of the code will be compiled with the basic protections of non-executable stacks. You must also be able to write a driver program to exploit these binaries then discover a method to escalate privileges. This will require advanced skills in binary exploitation to include the latest debugging concepts and egg hunting techniques. You must craft input code first to take control of program execution and second to map an area in memory to get your shellcode to work and bypass system protections.

Cloud Penetration Testing

The adoption of cloud computing in organizations is growing rapidly, which is why your ability to perform a cloud penetration test must be at it’s best! Checking for compliance and governance issues, checking if strong authentication is deployed for every remote user, checking data retention policy of service providers, performing automated cloud security testing, as well as AWS, Azure, and Google Cloud Platform penetration testing are just some of the thing you will be expected to be able to perform.

Wireless Penetration Testing

You will also be tested on your ability to perform WLAN penetration testing by discovering the wireless networks, detecting hidden SSIDs, sniffing wireless traffic, performing various attacks on the wireless network, cracking WEP, WPA, WPA2 keys, checking for MAC filtering, as well as penetrating RFID and Near Field Communication (NFC) systems.

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EC-Council was formed in 2001 after very disheartening research following the 9/11 attack on the World Trade Center. Founder and CEO of the EC-Council Group, Jay Bavisi, after watching the attacks unfold, raised the question, what if a similar attack were to be carried out on the cyber battlefield? Read More…

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LPTLow Platform Trailer

LPTLocal Property Tax (Ireland)

LPTLanguage Proficiency Test (various locations)

LPTLine Printer

LPTLicensed Professional Teacher

LPTLife Pro Tips

LPTLine Printer Terminal

LPTLow Pressure Turbine

LPTLicensed Physical Therapist

LPTLinear Position Transducer (medical equipment)

LPTLicensed Penetration Tester

LPTLight Pressure Turbo (automotive)

LPTLaw Practice Today

LPTLower Paxton Township (Pennsylvania)

LPTListed Property Trust

LPTLondon Probation Trust (UK)

LPTLicensed Psychiatric Technician

LPTLower-Order Path Termination

LPTLongest Processing Time (operations management)

LPTLocal Printer Terminal

LPTLimited Performance Test

LPTLocal Positioning Technology

LPTLoss Portfolio Transfer (financial reinsurance)

LPTLow Power Transceiver

LPTLow Profile Turret (gun)

LPTLunar Propellant Tanker

LPTLorain Palace Theater (Lorain, OH)

LPTLimited Private Type

LPTLoss Prevention Team

LPTLogistics Preparation of the Theater

LPTLiquid Penetrant Test (non destructive testing)

LPTLimited Phlebotomy Technician (licensed medical assistant)

LPTLinear Planarization Technology (semiconductors)

LPTLength-Preserving Transform (lossless text transformation)

LPTLogistic Plug Trunk

LPTLaser Propulsion Test

LPTLovelock Paiute Tribe (Nevada)

LPTLabor Process Theory

LPTLoad Port Transfer

LPTLamina Propria T Cell

LPTLow Pressure Test

LPTLimited Production Test

LPTLegal Professional Training

LPTLouisville's Play Toy (Matt Hayes)

LPTLandings Per Tire (aviation)

LPTLimited Power Technician (Minnesota State Board of Electricity license)

LPTLow Probability Targeting

LPTLocal Platform Testing (Sprint)

LPTLow Power Technique

LPTLow Profile Transformer

LPTLiterary Present Tense (language use)

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Tá an chuid seo den suíomh idirlín ar fáil i mBéarla amháin i láthair na huaire.

This section of the site is currently only available in English

Gaeilge

myAccount |

ROS

LPT Online

or LPT Online

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Home Property Local property tax

Local Property Tax (LPT)

LPT is a self-assessed tax charged on the market value of residential properties in the State.

基于BLI技术的脂多糖转运蛋白LptA/LptC相互作用检测方法的建立

生物工程学报 2021, Vol. 37 Issue (9): 3300-3309

http://dx.doi.org/10.13345/j.cjb.200709

中国科学院微生物研究所、中国微生物学会主办

文章信息

代晓伟, 朱小红, 司书毅, 李妍, 袁丽杰

Dai Xiaowei, Zhu Xiaohong, Si Shuyi, Li Yan, Yuan Lijie

基于BLI技术的脂多糖转运蛋白LptA/LptC相互作用检测方法的建立

Development of a BLI assay-based method for detecting LptA/LptC interaction

生物工程学报, 2021, 37(9): 3300-3309

Chinese Journal of Biotechnology, 2021, 37(9): 3300-3309

10.13345/j.cjb.200709

文章历史

Received: November 4, 2020

Accepted: February 5, 2020

Published: February 23, 2021

Abstract

PDF

Figures

Tables

引用本文

代晓伟, 朱小红, 司书毅, 等. 基于BLI技术的脂多糖转运蛋白LptA/LptC相互作用检测方法的建立. 生物工程学报, 2021, 37(9): 3300-3309

Dai XW, Zhu XH, Si SY, et al. Development of a BLI assay-based method for detecting LptA/LptC interaction. Chinese Journal of Biotechnology, 2021, 37(9): 3300-3309.

基于BLI技术的脂多糖转运蛋白LptA/LptC相互作用检测方法的建立

代晓伟1

朱小红2

司书毅2

李妍2

袁丽杰1

1. 华北理工大学基础医学院河北省慢性疾病基础医学重点实验室唐山市慢性病临床基础研究重点实验室，河北唐山 063210;

2. 中国医学科学院北京协和医学院医药生物技术研究所国家新药 (微生物) 筛选实验室，北京 100050

收稿日期：2020-11-04；接收日期：2020-02-05；网络出版时间：2021-02-23

基金项目：中国医学科学院医学与健康科技创新工程(No. 2016-12M-3-014)，国家科技重大专项重大新药创制(No. 2019ZX09721001-004-006)，河北省自然科学基金(No. H2021209027) 资助

摘要：革兰阴性菌脂多糖转运蛋白(Lipopolysaccharide transport，Lpt) LptA和LptC通过稳定的相互作用对脂多糖装配起到重要作用，它们相互作用的阻断会导致脂多糖层缺损和菌体死亡，因此具备成为抗菌药物筛选靶标的可行性。文中应用生物膜干涉(Biolayer interferometry，BLI) 技术对LptA/LptC相互作用进行检测，为建立体外的LptA/LptC蛋白相互作用阻断剂筛选方法奠定基础。首先在大肠杆菌Escherichia coli BL21(DE3) 中进行大肠杆菌LptA全长、LptA去信号肽和LptC蛋白的表达；纯化的蛋白使用生物素标记后结合到预先稀释液封闭的超级链霉亲和素(Super streptavidin，SSA) 生物传感器，然后再检测与未标记蛋白之间的结合信号，同时做无蛋白的稀释液对照；使用同样的方法检测生物素化蛋白与小分子的结合以及相互作用的阻断；空白对照采用未结合生物素化蛋白的传感器，检测上述系列稀释样品。响应信号采用稳态分析(Steady state analysis) 方式拟合，计算样品平衡常数(KD) 值。本研究成功获得高纯度的LptA和LptC蛋白，并且检测到结合传感器的LptC蛋白与LptA全长蛋白和LptA去信号肽蛋白均具有良好的结合活性，KD值分别为2.9e-7±7.9e-8、6.0e-7±2.8e-8；结合传感器的LptA去信号肽蛋白与LptC蛋白具有良好的结合活性，KD值为9.6e-7±7.2e-9；所有结合曲线呈现出明显的快结合-快解离形态。小分子化合物IMB-881能够与LptA结合来阻断LptA/LptC之间的相互作用，与LptC之间无结合活性。文中首次建立了基于BLI技术的LptA/LptC相互作用检测方法，并且证实该方法能够用于小分子阻断剂阻断活性的评价，为后续的LptA/LptC蛋白相互作用阻断剂筛选奠定了基础。

关键词：生物膜干涉脂多糖脂多糖转运蛋白 LptA LptC LptA/LptC相互作用

Development of a BLI assay-based method for detecting LptA/LptC interaction

Xiaowei Dai1

Xiaohong Zhu2

Shuyi Si2

Yan Li2

Lijie Yuan1

1. Tangshan Key Laboratory for Preclinical and Basic Research on Chronic Disease, Hebei Key Laboratory for Chronic Diseases, School of Basic Medical Science, North China University of Science and Technology, Tangshan 063210, Hebei, China;

2. National Center for Drug (Microbiology) Screening Laboratory, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

Received: November 4, 2020; Accepted: February 5, 2020; Published: February 23, 2021

Supported by: CAMS Initiative for Innovative Medicine (No. 2016-12M-3-014), National Mega-Project for Innovative Drugs (No. 2019ZX09721001-004-006), Natural Science Foundation of Hebei Province, China (No. H2021209027)

Corresponding author:

Yan Li. Tel: +86-10-63180623; E-mail: 13391513480@163.com;Lijie Yuan. Tel: +86-315-8805507; E-mail: yuanlijie1970@163.com.

Abstract: In Gram-negative bacteria, lipopolysaccharide transport (Lpt) protein LptA and LptC form a complex to transport LPS from the inner membrane (IM) to the outer membrane (OM). Blocking the interaction between LptA and LptC will lead to the defect of OM and cell death. Therefore, Lpt protein interaction could be used as a target to screen new drugs for killing Gram-negative bacteria. Here we used biolayer interferometry (BLI) assay to detect the interaction between LptA and LptC, with the aim to develop a method for screening the LptA/LptC interaction blockers in vitro. Firstly, LptC and LptA with or without signal peptide (LptAfull or LptAno signal) were expressed in E. coli BL21(DE3). The purified proteins were then labeled with biotin and the super streptavidin (SSA) biosensor was blocked with diluent. The biotin labeled protein sample was mixed with the sensor, and then the binding of the protein with a series of diluted non biotinylated protein was detected. At the same time, non-biotinylated protein was used as a control. The binding of biotinylated protein to a small molecule IMB-881 and the blocking of interaction were also detected by the same method. In the blank control, the biosensor without biotinylated protein was used to detect the serially diluted samples. The signal response constant was calculated by using steady analysis. The results showed that biotinylated LptC had a good binding activity with LptAfull and LptAno signal with KD value 2.9e-7±7.9e-8 and 6.0e-7±2.8e-8, respectively; biotinylated LptAno signal had a good binding activity with LptC, with a KD value of 9.6e-7±7.2e-9. All binding curves showed obvious fast binding and fast dissociation morphology. The small molecule compound IMB-881 can bind to LptA to block the interaction between LptA and LptC, but has no binding activity with LptC. In summary, we developed a method for detecting the LptA/LptC interaction based on the BLI technology, and confirmed that this method can be used to evaluate the blocking activity of small molecule blockers, providing a new approach for the screening of LptA/LptC interaction blockers.

Keywords:

biolayer interferometry (BLI) lipopolysaccharide (LPS) lipopolysaccharide transport (Lpt) LptA LptC LptA/LptC interaction

革兰阴性菌(G-菌) 感染率近年来逐年增高，已经成为临床细菌感染的主要病原菌[1-2]。耐药细菌的不断出现与传播使得细菌感染的预防与治疗面临严峻的挑战，因药物作用靶标发生改变而引起的耐药是主要耐药机制之一，因此发现新靶标，进而发现新型抗生素对抗菌药物的研发具有重要的意义。

脂多糖(Lipopolysaccharide，LPS) 是革兰阴性菌特有的结构，是菌体抵御外界环境的重要屏障，也是革兰阴性菌固有耐药的重要因素，因此LPS作为抗革兰阴性菌药物研发的靶标近年来受到越来越多的关注[3]。LPS在细胞内膜内侧合成后，经由脂多糖转运蛋白复合体(Lipopolysaccharide transport，Lpt) 转运到外膜进行组装，形成完整的LPS层[4-5]。Lpt复合物由7种蛋白(Lpt A-G)组成，其中位于内膜的LptC蛋白能够结合LPS，然后依靠内膜蛋白LptBFG提供的能量，将LPS传递给周质蛋白LptA，进而传递给外膜蛋白LptD和LptE进行组装，通过这种桥链作用完成LPS的转运和装配，最终形成LPS层[6-8]。LptA能够结合LPS，它与LptC蛋白和LptDE复合体也能形成稳定的相互作用，这种相互作用保证了LptA蛋白稳定地存在于周质空间，并且完成LPS的转运作用[9]。Lpt蛋白相互作用的阻断会破坏LPS的跨膜转运，进而影响外膜的生成，导致细菌死亡[10-11]。此外，Lpt蛋白具有较高的保守性，在哺乳动物细胞中也并不存在，目前尚未有以Lpt蛋白为靶标的抗菌药物应用于临床，因此Lpt蛋白相互作用具有成为抗革兰阴性菌药物发现靶标的潜力[12-13]。

生物膜干涉(Biolayer interferometry，BLI) 技术是一种光干涉技术和光纤信号测量技术相结合的光学分析技术，分子与分子之间的特异性作用会改变传感器表面生物膜的厚度，从而形成光谱位移值。因此，该技术可以实时检测蛋白和小分子之间或者蛋白之间的相互作用，通过平衡常数(KD) 来表示相互作用的强弱。该技术近年来在抗体筛选、配体受体研究以及蛋白与小分子结合方面多有应用[14]。

鉴于LptA/LptC蛋白相互作用在革兰阴性菌中的重要作用，本研究建立了基于BLI技术的LptA/LptC蛋白相互作用的检测方法，并在此基础上检测已报道的阻断剂IMB-881的阻断活性，以进一步评价BLI技术能否应用于LptA/LptC蛋白相互作用阻断剂的筛选，也为建立基于BLI技术的LptA/LptC蛋白相互作用阻断剂筛选模型和阻断活性评价模型奠定基础，推动Lpt蛋白相互作用作为抗菌药物筛选靶标的研究。

1 材料与方法

1.1 材料

1.1.1 试剂 IMB-881由中国医学科学院医药生物技术研究所国家新药(微生物) 筛选实验室合成；蛋白标准分子量购自BioRad公司；XhoⅠ、BamHⅠ购自TaKaRa公司；蛋白胨、酵母粉、琼脂粉购自Clontech公司；氨苄西林、卡那霉素、异丙基-β-D-硫代半乳糖苷(Isopropyl-β-D-thiogalactoside，IPTG) 购自Aladdin公司；免疫印迹膜、HisTrapTM层析柱购自GE公司；BCA (Bicinchoninic acid) 蛋白质定量试剂盒购自Thermo公司；小鼠抗组氨酸(Histidine，His) 标签单克隆抗体、辣根过氧化物酶(Horseradish peroxidase，HRP) 标记的羊抗小鼠IgG购自康为世纪公司；HRP底物化学发光液(ECL) 购自美国Millipore公司；96孔板购自Corning Costar公司；磷酸盐缓冲液(Phosphate buffered solution，PBS) 购自北京中杉金桥生物技术有限公司；吐温-20 (Tween-20) 购自Sigma公司；生物素化标记试剂NHS-LC-LC-Biotin购自Thermo公司；牛血清白蛋白(Bovine serum albumin，BSA)购自索莱宝公司；Zeba desalting spin columns脱盐柱购自Thermo公司；SSA传感器购自ForteBio公司；pET-28a表达载体由本实验室保存；其他试剂均为国产分析纯。

1.1.2 仪器超净工作台(哈东联Clean Benah公司)；加热制冷型金属浴(Thermo公司)；高速冷冻离心机(Hitachi公司)；蛋白质电泳仪(GE公司)；半干转膜仪(Bio-Rad公司)；恒温摇床(北京沃德创技贸有限公司)；超声破碎仪(B.Braun Biotech International公司)；AKTA层析系统(GE公司)；凝胶成像系统(上海天能公司)；多功能微孔板检测仪(PerkinElmer公司)；分子相互作用检测仪器(Octet Red96，美国Pall公司)。

1.2 方法

1.2.1 LptA和LptC蛋白的原核表达和纯化在NCBI数据库中检索LptA基因(GenBank登录号：QBC14848.1) 和LptC基因(GenBank登录号：QBC14847.1)，然后对Lpt基因序列进行GC含量计算与基因序列优化，优化的序列中避免XhoⅠ与BamHⅠ酶切位点。在优化的LptA、LptC基因序列5′端和3′端分别加入XhoⅠ与BamHⅠ酶切位点进行基因合成。构建重组质粒pET-28a-LptA全长、pET-28a-LptA去信号肽和pET-28a-LptC，以热激法转化到大肠杆菌Escherichia coli BL21(DE3) 感受态细胞中，再以卡那霉素抗性筛选法进行阳性重组子筛选。随机挑取重组子接种到含30 μg/mL卡那霉素抗性的LB液体培养基中，37 ℃培养。当OD值达到0.6时，添加0.5 mmol/L诱导剂IPTG，继续培养。分别于20 ℃条件下培养过夜或37 ℃条件下培养6 h诱导表达，未添加诱导剂的为阴性对照，再以12% SDS-PAGE进行分析。

为了获得纯化的蛋白，将诱导后的菌体(0.5 mmol/L IPTG，20 ℃，过夜) 以超声波法破碎后，离心收集上清。按照HisTrapTM亲和层析法进行蛋白分离纯化。纯化的蛋白分别以0.2% 月桂酰肌氨酸钠(Sarkosyl，SKL) 溶液透析和蛋白质浓缩超滤管浓缩，0.45 μm滤膜过滤后以BCA法定量，应用蛋白质印迹分析方法检测蛋白的正确表达。

1.2.2 BLI技术检测蛋白间相互作用 1) 样品制备：参照说明书对准备结合到传感器的LptA和LptC蛋白进行生物素化，LptA全长、LptA去信号肽和LptC浓度分别为0.6 mg/mL、0.8 mg/mL和0.24 mg/mL，生物素浓度1 mmol/L，生物素化的蛋白使用脱盐柱处理后，使用PBST (含有0.02%吐温-20的PBS) 缓冲液稀释至25 μg/mL。

2) 蛋白相互作用检测：4根SSA传感器分别置于传感器支架第一、二列，每列两根，第一列结合生物素化蛋白，第二列为空白对照。待测样品使用PBST缓冲液自25 μg/mL起2倍梯度稀释至浓度为1.56 μg/mL。在96孔板中按照表 1所示加入样品，A行为待测样品，将对照行B的样品孔替换为等量的PBST缓冲液，其余各孔不变。

在Data Acquisition 9.0软件中将检测条件设置如下：传感器在第一孔PBS浸润10 min之后，按照Baseline (60 s)、Loading (600 s)、Wash (60 s)进行生物素化蛋白的结合，然后按照Baseline (60 s)、Association (90 s) 和Dissociation (90 s) 检测不同样品孔与传感器的结合。

1.2.3 BLI技术检测蛋白与IMB-881的相互作用检测LptA全长、LptA去信号肽、LptC分别与小分子IMB-881的相互作用，SSA传感器的预处理及检测方法与1.2.2相同。使用PBST-BSA (含有0.1% BSA的PBST，pH 6.0) 缓冲液将IMB-881化合物自38.6 μmol/L浓度起梯度稀释至0.15 μmol/L，表 1中的S换成稀释好的IMB-881化合物，R为PBST-BSA缓冲液。

表 1 样品排布

Table 1 Sample arrangement

B: buffer, PBS; L: loading, biotinylated protein; S: sample; R: reference, PBST.

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1.2.4 BLI技术检测IMB-881对蛋白的阻断作用 SSA传感器的预处理及检测与1.2.2中相同。但是表 1中的S换成均由PBST-BSA缓冲液稀释的IMB-881化合物和蛋白的混合液，R为PBST-BSA缓冲液。

1.2.5 数据分析使用Data analysis 9.0软件对数据进行分析。以对应的未结合蛋白的传感器及缓冲液对照作为对照进行扣除，以稳态分析(Steady state analysis)方式拟合，如R2≥0.95，则检测有效，计算响应值与KD值。

2 结果与分析

2.1 LptA和LptC基因的优化基因序列的优化有利于提高其在大肠杆菌中的表达量[15]，在本研究中应用SnapGene和DNAMAN软件分析，LptA全长原始序列的GC含量为49%，经优化后GC含量为50%且均匀分布；LptA去信号肽原始序列的GC含量为50%，经优化后GC含量为49%，但均匀分布；LptC原始序列的GC含量为47%，经优化后GC含量为51%且均匀分布(图 1)。

图 1 LptA全长、LptA去信号肽及LptC基因优化前后的序列比较

Fig. 1 Comparison of the sequences of LptAfull, LptAno signal and LptC before and after optimization.

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2.2 LptA和LptC原核表达质粒的构建与鉴定信号肽对于蛋白的跨膜定位具有重要的作用，蛋白表达中也发现带有信号肽的蛋白更容易以可溶性蛋白形式表达[16]，但是在周质空间中的LptA蛋白是切除了信号肽的蛋白，信号肽可能会影响蛋白间相互作用。为了获得可溶性的活性蛋白，而且不影响蛋白之间的相互作用，同时表达了带有信号肽的蛋白(LptA全长) 和去掉信号肽的蛋白(LptA去信号肽)。

因而在LptA构建策略中，分别构建氨基端含信号肽与去信号肽的重组质粒。将优化的基因合成后，以XhoⅠ和BamHⅠ为酶切位点，连接到pET-28a表达载体中构建重组质粒pET-28a-LptA全长、pET-28a-LptA去信号肽、pET-28a-LptC，羧基端仅融合多聚组氨酸标签。重组质粒以双酶切法进行鉴定，得到了与预期大小(550 bp) 一致的LptA全长基因片段(图 2A)，同样方法得到预期大小(489 bp) 一致的LptA去信号肽基因片段(图 2B)。在LptC构建策略中，构建羧基端带有融合多聚组氨酸标签的无信号肽蛋白重组质粒。重组质粒以双酶切法进行鉴定，得到了与预期大小(483 bp) 一致的LptC基因片段(图 2C)。上述序列经测序比对分析显示，其基因序列与预期优化序列完全一致，氨基酸序列与天然Lpt一级结构完全相同，表明成功构建了重组质粒pET-28a-LptA全长、pET-28a-LptA去信号肽和pET-28a-LptC。

图 2 LptA全长、LptA去信号肽及LptC原核表达质粒的酶切鉴定

Fig. 2 Enzymatic identification of LptAfull, LptAno signal and LptC prokaryotic expression plasmid. 1: DNA marker; 2: gene.

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2.3 LptA和LptC原核表达及纯化卡那霉素抗性筛选的重组子分别在20 ℃和37 ℃，1 mmol/L的IPTG诱导表达，经由SDS-PAGE分析发现在22 kDa、19 kDa和20 kDa均有明显条带，LptA蛋白在沉淀中，LptC蛋白在上清中(图 3A)。

图 3 LptA、LptC原核表达及纯化检测

Fig. 3 Prokaryotic expression and purification of LptA and LptC. (A) Protein-induced expression. LptAfull and LptC: M: protein marker; 1: total cell protein before induction; 2: cellular lysate supernatant at 20 ℃; 3: inclusion body at 20 ℃; 4: cellular lysate supernatant at 37 ℃; 5: inclusion body at 37 ℃; LptAno signal: M: protein marker; 1: total cell protein before induction; 2, 4: cellular lysate supernatant at 20 ℃; 3, 5: inclusion body at 20 ℃; 6: cellular lysate supernatant at 37 ℃; 7: inclusion body at 37 ℃. (B) Analysis of the purified Lpt using SDS-PAGE analysis. (C) Analysis of the purified Lpt using Western blotting assay.

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纯化后的蛋白经由SDS-PAGE分析，结果表明，纯化的2种LptA蛋白和LptC蛋白经考马斯亮蓝染色后，分别在预期分子量22 kDa、19 kDa和20 kDa位置呈单一条带，说明纯化的3种蛋白具有较高的纯度(图 3B)。蛋白质印迹分析实验结果显示，在相同分子量位置仍有单一条带，证实了3种蛋白的正确表达(图 3C)。纯化的LptA全长、LptA去信号肽和LptC蛋白经透析和浓缩后，其浓度分别为0.80 mg/mL、0.60 mg/mL和0.24 mg/mL。

2.4 BLI技术检测蛋白间的相互作用对两种LptA蛋白与LptC蛋白之间的相互作用分别进行了检测(图 4)。结果发现，传感器结合生物素化的LptC蛋白时，LptC与2个LptA蛋白均能够结合，LptA蛋白从3.125 μg/mL上升至25 μg/mL时，LptC与LptA全长的信号值从0.046 3上升至0.101 3，与LptA去信号肽的信号值从0.027 5上升至0.080 9，具有浓度依赖性。传感器结合生物素化的LptA全长或LptA去信号肽时，也能检测到两个蛋白与LptC的相互作用，随着LptC浓度从3.125 μg/mL上升至12.5 μg/mL，LptA全长响应值从0.001 2上升至0.016 0，LptA去信号肽的信号值则由0.025 9上升至0.082 6，均具有浓度梯度依赖性。

图 4 LptA/LptC相互作用检测

Fig. 4 Detection of LptA/LptC interaction. (A) The interaction between LptC protein and LptAfull protein. (B) The interaction between LptC protein and LptAno signal protein. (C) The interaction between LptAfull protein and LptC protein. (D) The interaction between LptAno signal protein and LptC protein.

图选项

此外，这些结果也表明信号肽是否存在对LptA和LptC蛋白的结合并没有明显影响。但是传感器在结合生物素化LptA全长时，检测值明显低于结合LptA去信号肽的值，这可能与LptA全长与传感器的结合能力较弱有关系，信号肽的存在可能会影响LptA蛋白生物素化或与传感器的结合。

2.5 BLI技术检测蛋白与小分子相互作用 IMB-881是已经报道的具有LptA/LptC相互作用阻断活性的小分子化合物[17]，该化合物能够通过与LptA结合阻断两个蛋白的相互作用。为了验证BLI技术是否能够应用于检测小分子化合物对相互作用的阻断，以IMB-881为阳性化合物进行了评价(图 5)。因为传感器结合LptA全长蛋白时，与LptC蛋白相互作用信号值比较弱，所以在IMB-881与蛋白的结合以及后面的阻断实验中，选用了LptA去信号肽蛋白。结果发现，IMB-881能够与LptA结合，并且响应值随着化合物浓度的升高而增加，具有浓度依赖性，在9.65 μmol/L和38.60 μmol/L时的响应值分别为0.020 2和0.037 7。IMB-88与LptC无相互作用，这与已经报道的结果相一致[17]。

图 5 蛋白与IMB-881相互作用的检测

Fig. 5 Detection of protein interaction with IMB-881. (A) The interaction between LptAno signal protein and IMB-881. (B) The interaction between LptC protein and IMB-881.

图选项

2.6 BLI技术检测小分子对蛋白的阻断作用为了验证IMB-881能够阻断LptA-LptC相互作用，将生物素化的LptA去信号肽固化到传感器上，使用不同浓度的IMB-881与LptC蛋白共孵育后流经进行检测。由于IMB-881与空传感器具有较强的非特异性结合，会掩盖相互作用的结果，在查阅相关文献[18]后，对缓冲液进行优化，使用PBST-BSA缓冲液对未结合蛋白的空传感器进行一定的封闭。结果如图 6所示，IMB-881能够阻断LptA-LptC相互作用，且具有浓度依赖性。

图 6 IMB-881对LptA-LptC相互作用阻断的检测

Fig. 6 Disruption of LptA-LptC protein interaction by IMB-881.

图选项

3 讨论作为革兰阴性菌特有的重要结构，近年来LPS在抗菌药物研发中受到越来越多的关注，以它的合成、跨膜转运以及在外膜的组装各个环节的关键调控因子为靶标的药物研究也多有报道[4, 19]，最早研究的LPS合成抑制相关的Lipid A合成关键酶LpxC抑制剂在体内外均表现出良好的抗菌活性[20]。随着LPS转运和组装机制的解析，以其中的关键因子为靶标的药物发现也多有研究[21-23]。

在2018年，Zhang等构建了LptA和LptC相互作用的酵母双杂交模型，并在此基础上发现了具有抗菌活性的IMB-881化合物，提示了该相互作用作为抗菌药物靶标的可行性[17]。基于酵母双杂交模型的筛选方法简单，能够实现高通量筛选，但是因为酵母细胞壁与细菌细胞壁的差异很大、化合物对酵母细胞本身也会产生毒性或者抑制报告基因的表达，从而导致漏筛和假阳性的产生。此外，筛选得到的阳性化合物也需要在分子水平上进一步的确认，基于表面等离子共振(Surface Plasmon Resonance，SPR) 技术建立了LptA/LptC相互作用的检测方法，该方法的灵敏度比较高，但是该方法通量低，8通道的仪器也只能做到同时对7个样本的检测，而且检测时间较长，很难实现高通量的相互作用评价和筛选。免疫共沉淀和拉下(Pull down) 等传统技术中因为蛋白质印迹分析技术的复杂操作，也很难应用于高通量的评价和筛选，因此，有必要建立一种简单的蛋白相互作用和阻断活性评价方法，以推动分子水平上的相互作用阻断剂的筛选和后续的阻断活性的鉴定。BLI技术是新一代检测生物分子相互作用的实时分析监测技术，由于其分析速度快、无标记、特异性强等优点而被广泛应用于快速检测[14, 24-25]。蛋白生物素化提高了蛋白与芯片的结合能力，提高了灵敏性，而96通道甚至384通道仪器的开发也使得该技术能够快速地同时测定多个蛋白相互作用或者同时评价蛋白与多个化合物的相互作用，使得高通量筛选成为可能。BLI技术在研究抗原/抗体相互作用[26]、蛋白与小分子化合物相互作用方面多有应用，但是在蛋白相互作用方面并不多见。本研究建立了基于BLI技术的LptA/LptC蛋白相互作用的检测方法，发现生物素化的LptA和LptC蛋白能够很好地结合到传感器上，能够检测到较高的相互作用信号，以IMB-881为阳性药物也能发现化合物与蛋白的结合和相互作用的阻断。这为后续的相互作用小分子的筛选和阻断活性的评价奠定了良好的基础，也推动了BLI技术在蛋白相互作用方面的应用。

在进行IMB-881阻断LptA-LptC相互作用时发现，IMB-881与空探针的非特异性结合较强，会掩盖阻断活性的结果。在本研究中，采用0.1% BSA封闭未结合蛋白的探针位点来消除非特异性结合。同时发现在检测相互作用阻断时，不同的蛋白结合到传感器上对阻断的结果影响也不相同。因此，直接应用两个蛋白进行相互作用筛选阻断剂是很困难的，因此在实际应用BLI技术进行相互作用筛选阻断剂时，更为可行的筛选策略应该是首先应用单一蛋白筛选与蛋白相互作用的小分子化合物，在得到阳性化合物后再进行阻断活性的复筛。

综上所述，本研究基于BLI技术实时检测分子间相互作用，建立优化的基于BLI技术检测小分子抑制剂阻断Lpt蛋白相互作用的方法，为后续的小分子抑制剂的筛选以及阻断活性的评价奠定了实验基础。

参考文献

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Simpson BW, Trent MS. Pushing the envelope: LPS modifications and their consequences. Nat Rev Microbiol, 2019, 17(7): 403-416. DOI:10.1038/s41579-019-0201-x

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Tran AX, Dong CJ, Whitfield C. Structure and functional analysis of LptC, a conserved membrane protein involved in the lipopolysaccharide export pathway in Escherichia coli. J Biol Chem, 2017, 292(45): 33529-33539.

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Schultz KM, Fischer MA, Noey EL, et al. Disruption of the E. coli LptC dimerization interface and characterization of lipopolysaccharide and LptA binding to monomeric LptC. Protein Sci, 2018, 27(8): 1407-1417. DOI:10.1002/pro.3429

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Schultz KM, Feix JB, Klug CS. Disruption of LptA oligomerization and affinity of the LptA-LptC interaction. Protein Sci, 2013, 22(11): 1639-1645. DOI:10.1002/pro.2369

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张艳, 姜丽艳, 张晓光, 等. 生物膜干涉技术在生物分子间相互作用检测中的应用. 生命科学仪器, 2019, 18(2): 49-52, 42. Zhang Y, Jiang LY, Zhang XG, et al. Application of biolayer interferometry in detection of biomolecular interaction. Life Sci Instrum, 2019, 18(2): 49-52, 42 (in Chinese).

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陈云雨, 付正豪, 闫干干, 等. 基于密码子优化策略的新型冠状病毒主蛋白酶在大肠杆菌中的表达条件优化与活性鉴定. 生物工程学报, 2020, 37(4): 1-12. Chen YY, Fu ZH, Yan GG, et al. Expression condition optimization and proteolytic activity analysis of codon-optimized SARS-CoV-2 main protease in Escherichia coli. Chin J Biotech, 2020, 37(4): 1-12 (in Chinese).

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郑屠园, 李娅慧, 周继勇, 等. 信号肽序列对A型流感病毒H1N1 HA蛋白表达的影响. 畜牧与兽医, 2020, 52(8): 64-67. Zheng TY, Li YH, Zhou JY, et al. Effect of signal peptide on the expression of the H1N1 HA protein of the influenza A virus. Animal Husband Vet Med, 2020, 52(8): 64-67 (in Chinese).

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张峰, 武刚, 于传飞, 等. 应用生物膜干涉技术测定治疗性单抗与新生儿Fc受体的亲和力. 中国新药杂志, 2016, 25(16): 1861-1867. Zhang F, Wu G, Yu CF, et al. Detection of affinity between therapeutic monoclonal antibodies and neonatal Fc receptor using biolayer interferometry. Chin J New Drugs, 2016, 25(16): 1861-1867 (in Chinese).

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Ho H, Miu A, Alexander MK, et al. Structural basis for dual-mode inhibition of the ABC transporter MsbA. Nature, 2018, 557(7704): 196-201. DOI:10.1038/s41586-018-0083-5

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Tomaras AP, McPherson CJ, Kuhn M, et al. LpxC inhibitors as new antibacterial agents and tools for studying regulation of lipid A biosynthesis in Gram-negative pathogens. mBio, 2014, 5(5): e01551-14.

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Vetterli SU, Zerbe K, Müller M, et al. Thanatin targets the intermembrane protein complex required for lipopolysaccharide transport in Escherichia coli. Sci Adv, 2018, 4(11): eaau2634. DOI:10.1126/sciadv.aau2634

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Moura ECCM, Baeta T, Romanelli A, et al. Thanatin impairs lipopolysaccharide transport complex assembly by targeting LptC-LptA interaction and decreasing LptA stability. Front Microbiol, 2020, 11: 909. DOI:10.3389/fmicb.2020.00909

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Srinivas N, Jetter P, Ueberbacher BJ, et al. Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa. Science, 2010, 327(5968): 1010-1013. DOI:10.1126/science.1182749

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杨国泰, 吴鑫, 李福来, 等. 生物膜层干涉技术在生物分子分析和检测中应用的研究进展. 分析测试学报, 2017, 36(8): 1055-1060. Yang GT, Wu X, Li FL, et al. Research progress in application of biolayer interferometry in analysis and detection of biomolecules. J Instrum Anal, 2017, 36(8): 1055-1060 (in Chinese). DOI:10.3969/j.issn.1004-4957.2017.08.020

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连接到 LPT 端口的打印机

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06/15/2023

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LPT 枚举器是总线驱动程序的一个示例。 LPT 枚举器能够从符合 IEEE 1284 扩展功能端口协议和 ISA 接口标准的 LPT 端口硬件获取标识信息。

Windows 2000 或更高版本系统启动时，配置管理器会调用 LPT 枚举器来枚举连接到 LPT 端口的 IEEE 1284 兼容设备。对于找到的每个设备，配置管理器将调用打印机类安装程序。打印机类安装程序调用 SetupDi 前缀设备安装函数，从打印机 INF 文件获取信息。

对于并行连接的打印机，并行枚举器使用从打印机接收的 1284 字符串生成的唯一硬件 ID 创建开发节点。

示例 1284 字符串为：

"MANUFACTURER:Hewlett-Packard;COMMAND SET:PJL,MLC,PCL,POSTSCRIPT;MODEL:HP Color LaserJet 550;CLASS:PRINTER;COMMENT:HP LaserJet;"

从此 1284 字符串中，并行枚举器生成以下硬件 ID：

LPTENUM\Hewlett-PackardHP_Co3115

硬件 ID 由枚举器前缀组成，后跟制造商名称、型号名称和循环冗余检查 (CRC) 代码。 CRC 代码（硬件 ID 的最后四位数字）是从制造商和型号字符串生成的。字符串中的空格将替换为下划线。

若要从设备读取 1284 ID 字符串，请发送 IOCTL_PAR_QUERY_DEVICE_ID。请注意，后台处理程序将 LPTx 符号链接重定向 (其中 x 是 LPT 数字 1、2 或 3) 到后台处理程序的命名管道，这意味着如果后台处理程序正在运行，则 parport 永远不会看到发送到 LPTx 的 IOCTL。

并行连接的即插即用打印机的开发节点放置在 HKLM\SYSTEM\CurrentControlSet\Enum\LPTENUM 下，并且具有以下形式的单个硬件 ID：

LPTENUM\Company_NameModelNam1234

驱动程序堆栈显示在下一个代码示例后面的图中。

以下示例显示了正确“即插即用”LPTENUM\Company_NameModelNam1234 形式的硬件 ID 的 INF 代码。请注意，“型号名称 XYZ”设备说明在 INF 制造商部分中出现两次。第一行中的硬件 ID 包括总线枚举器，而第二行中的硬件 ID 不包含。无论安装了打印机的总线类型如何，这两行都保证排名 0 的硬件 ID 匹配。有关详细信息，请参阅安装自定义即插即用打印机驱动程序。

[Manufacturer]

%Company_Name%=Company_Name

; Section name for all drivers for Company_Name

[Company_Name]

"Model Name XYZ" = Install_Section_XYZ, LPTENUM\Company_NameModelNam1234 ; plus any compatible IDs

"Model Name XYZ" = Install_Section_XYZ, Company_NameModelNam1234 ; plus any compatible IDs

; The install section for the XYZ model

[Install_Section_XYZ]

[Strings]

Company_Name="Company Name"

对于与其他型号共享其设备 ID 的打印机，INF 文件应如下所示：

[Manufacturer]

%Company_Name%=Company_Name

; The section for all drivers for Company_Name

[Company_Name]

"Model Name XYA" = Install_Section_XYA, LPTENUM\Company_NameModelNam1234, Company_NameModelNam1234 ; plus any other compatible IDs

"Model Name XYA" = Install_Section_XYA, Company_NameModelNam1234, Company_NameModelNam1234 ; plus any other compatible IDs

"Model Name XYB" = Install_Section_XYB, LPTENUM\Company_NameModelNam1234, Company_NameModelNam1234; plus any other compatible IDs

"Model Name XYB" = Install_Section_XYB, Company_NameModelNam1234, Company_NameModelNam1234 ; plus any other compatible IDs

; The install sections

[Install_Section_XYA]

[Install_Section_XYB]

[ControlFlags]

InteractiveInstall = LPTENUM\Company_NameModelNam1234, Company_NameModelNam1234

[Strings]

Company_Name = "Company Name"

与前面的示例一样， INF Manufacturer 部分中的每个模型都由一对几乎相同的线条表示。对于给定模型，对中的一行包括总线枚举器;另一个没有。无论安装了打印机的总线类型如何，这两行都保证排名 0 的硬件 ID 匹配。有关详细信息，请参阅安装自定义即插即用打印机驱动程序。

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