CN111996212B - 序列的应用、重组载体及其提高pvx植物表达载体蛋白表达量的方法 - Google Patents
序列的应用、重组载体及其提高pvx植物表达载体蛋白表达量的方法 Download PDFInfo
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Abstract
本发明涉及植物生产重组蛋白领域，特别涉及序列的应用、重组载体及其提高PVX植物表达载体蛋白表达量的方法。发现CP基因3`端至少50bP的序列对提高目的基因的表达有明显的作用，序列详见表1。在目的基因开放阅读框后保留CP基因3`端50bp的序列，能显著提高目的基因的表达。
Description
技术领域
本发明涉及植物生产重组蛋白领域，特别涉及序列的应用、重组载体及其提高PVX植物表达载体蛋白表达量的方法。
背景技术
随着生物医药技术和产业的不断发展，预计生物制品市场份额将从2016年的25％(2020亿美元)上升到2022年的30％(3620亿美元)。重组蛋白表达式生物制品生产的一个重要组成部分，通过不同的表达平台将目标蛋白大量、稳定的表达并具有相应的生物学功能，最终应用于临床或其他领域。目前，常见蛋白表达平台包括：大肠杆菌、酿酒酵母、杆状病毒/昆虫细胞、哺乳动物细胞等，不同表达平台有各自的优点和缺点。当前，绝大多数生物制药的表达平台是哺乳动物细胞和酵母。但哺乳动物细胞表达成本高，表达量相对较低；酵母表达蛋白折叠和修饰有一定的限制，需要寻求其他新型的表达系统以更便宜、更大量得表达合适修饰和折叠的重组蛋白。
植物是一种光合自养生物，其生长培育成本低、易于大规模培养、基因操作简单、蛋白质在植物体内能正确的折叠且不含对人类有害的病毒，是一种非常有潜力的蛋白表达平台。目前，植物蛋白表达平台主要包括转基因植物稳定表达和植物瞬时表达两种。转基因植物生长周期长且受到国家法规的严格控制，大大限制了其在蛋白表达中的应用。随着科学的发展和技术的进步，植物瞬时蛋白表达平台在蛋白表达中的应用越来越广。植物瞬时表达将目标蛋白表达框插入到T-DNA中，借助农杆菌将T-DNA导入植物细胞中表达，从机理上可以分为：病毒型(virus-based vector)和非病毒型(non-virus-based vector)。病毒型瞬时表达利用植物病毒在植物细胞快速增殖的特征，可以大大提高目标蛋白的表达和积累。目前，烟草花叶病毒(Tobacco Mosaic Virus,TMV)、马铃薯X病毒(Potato Virus X，PVX)和菜豆黄矮病毒(Bean Yellow and Dwarf Virus，BeYDV)等病毒被改造用于植物的瞬时表达用于重组蛋白的表达和生产。
PVX是一种单链正义RNA病毒，基因组约6.4kb。病毒基因组包含5个开放表达框，编码5个蛋白：ORF1编码RNA依赖的RNA聚合酶(RNA-dependent RNA Polymerase，RDRP)，三个重叠开放阅读框(ORF2、PRF3&ORF4)组成的移动蛋白(Movement Protein，MP)和ORF5编码的外壳蛋白(Coat Protein，CP)(图1)(Hull,2002)。PVX载体相关原件被改造用于植物外源基因的表达，现阶段一般作法是使用花椰菜花叶病毒35S启动子驱动PVX病毒RDRP的表达，保留PVX病毒亚基因组启动子(subgenomic promoter)用于驱动目的基因的表达，目的基因后连接终止子(Mardanova et al.,2017)。另外有其他研究表明PVX病毒CP蛋白对PVX载体的稳定性有重要作用(Chapman,Kavanagh,&Baulcombe,1992)。
发明内容
有鉴于此，本发明针对当前PVX植物表达载体进行改进，发现添加一些序列能够显著提高目的基因的表达，提高重组蛋白的产量。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了CP基因的3`端至少50bP的序列在促进PVX植物表达载体中外源目的基因的表达的应用。
在本发明的一些具体实施方案中，所述序列如SEQ ID No.1～3中任一项所示。
在本发明的一些具体实施方案中，所述外源目的基因包括GFP基因。
此外，本发明还提供了CP基因的3`端至少50bP的序列在提高PVX植物表达载体中蛋白表达量的应用。在本发明的一些具体实施方案中，所述序列如SEQ ID No.1～3中任一项所示。在本发明的一些具体实施方案中，所述蛋白包括GFP。
在上述研究的基础上，本发明还提供了重组载体，包括PVX植物表达载体、外源目的基因以及CP基因的3`端至少50bP的序列。在本发明的一些具体实施方案中，所述序列如SEQ ID No.1～3中任一项所示。
本发明还提供了所述的重组载体在提高外源目的基因的表达或蛋白表达量中的应用。
本发明海提改娥提高PVX植物表达载体蛋白表达量的方法，构建如权利要求7或8所述的重组载体，转染农杆菌，培养，提取蛋白。
本发明研究了CP蛋白序列对PVX载体外源目的基因表达的作用，最终发现CP基因3`端至少50bP的序列对提高目的基因的表达有明显的作用，序列详见表1。在目的基因开放阅读框后保留CP基因3`端50bp的序列，能显著提高目的基因的表达。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示PVX基因结构示意图；
图2示文献中PVX表达载体；
图3示本发明中构建PVX载体；
图4示不同PVX载体GFP表达情况对比；
图5示不同载体GFP表达Western-blot检测。
具体实施方式
本发明公开了序列的应用、重组载体及其提高PVX植物表达载体蛋白表达量的方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明以GFP为目标蛋白验证CP蛋白序列对PVX载体的蛋白表达量的作用。首先，根据实验设计构建表达载体(图3)，PVX-ori参照上述文献构建，PVX-CP200在GFP终止密码子后添加CP基因3`端200bp的序列(表1CP-200)，PVX-CP100在GFP终止密码子后添加CP基因3`端100bp的序列(表1CP100)，PVX-CP50在GFP终止密码子后添加CP基因3`端50bp的序列(表1CP50)。PVX CP基因序列参照NCBI登陆号MK387315.1，AF528555.1，EU031437.1,MK587458.1等序列。农杆菌双元载体使用pCambia1300，后续将各载体转入农杆菌GV3101中。培养农杆菌，并使用注射器进行烟草瞬时转化。注射后5天，观察GFP荧光并提取烟草蛋白质进行Western-blot分析。
结果如图4和5及表2所示，在目标基因后添加CP蛋白能著提高GFP的表达，PVX-CP200、PVX-CP100和PVX-CP50载体GFP表达量是PVX-ori的3倍以上，差异极显著(p<0.01)，而PVX-CP200、PVX-CP100和PVX-CP50载体GFP之间GFP表达无明显差异。
在当前PVX植物表达载体研究基础上，本发明发现了一种能够明显提高PVX载体蛋白表达的方法。此发明涉及添加PVX病毒一段CP序列到目标基因后，在不影响其他操作的基础上，大大提高了重组蛋白的表达量，降低植物重组蛋白生产的成本。
本发明提供的序列的应用、重组载体及其提高PVX植物表达载体蛋白表达量的方法中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
以pCambia1300为基础，根据实验需求构建了4个载体(图3)，其中PVX-ori参照当前常见PVX载体构建；PVX-CP200在GFP终止密码子后添加CP基因3`端250bp的序列，PVX-CP100在GFP终止密码子后添加CP基因3`端100bp的序列，PVX-CP50在GFP终止密码子后添加CP基因3`端50bp的序列。
表1 PVX CP序列
实施例2
将实施例1制备的各个载体使用电击转化法转化农杆菌GV3101。使用YEP培养基培养农杆菌至OD600达到3.0，6000rpm离心10分钟收集菌体。使用MES缓冲液(10mM MES，10mMMgCl2，100μM As)重悬菌体后6000rpm离心10分钟收集菌体。最后使用MES缓冲液稀释农杆菌至OD600至0.8。将稀释好的农杆菌使用注射器进行烟草浸润，每一个叶片右边为PVX-ori作为对照，左边为添加不同长度CP基因的载体。
实施例3
转化后植物在28℃，16：8光照比，培养5天后，进行GFP观测并提取叶片蛋白进行Western blot分析。Western-blot分析中，称取0.1g叶片，液氮研磨，之后加入200μL PBS缓冲液重悬，并在涡旋振荡器中平衡5分钟，12000rpm离心5分钟，取上清制备蛋白样品进行Western-blot分析。Western-blot首先使用8％SDS-PAGE进行分离，后续将蛋白转移至PVDF膜上。使用5％脱脂牛奶进行封闭1小时，后使用GFP特异性抗体孵育1小时，之后使用二抗孵育1小时后，使用DAB显色。
实施例4
对叶片蛋白提取液进行GFP ELISA定量检测，检测使用武汉云克隆科技股份有限公司绿色荧光蛋白(GFP)检测试剂盒(酶联免疫吸附实验法)进行：将叶片蛋白提取物使用PBS缓冲液稀释50倍后加样100μL，37℃孵育1小时；吸去上清废液，加入检测溶液A 100μL，37℃孵育1小时；洗板三次；加入检测溶液B 100μL，37℃孵育30分钟；洗板5次；加入TMB底物90μL，37℃孵育20分钟；加入终止液，在450nm波长下检测吸光值。每个载体采集3个样本，不同基因间检测结果比较使用student t-检测进行分析。
效果例
结果如图4和5及表2所示，在目标基因后添加CP蛋白能著提高GFP的表达，相较于PVX-ori，PVX-CP200、PVX-CP100和PVX-CP50载体GFP表达了明显提高，而PVX-CP200、PVX-CP100和PVX-CP50载体GFP之间，GFP表达无明显差异。最后，使用GFP ELISA定量检测试剂盒对叶片上清中GFP进行定量检测分析，最终结果与Western-blot相似，PVX-CP200、PVX-CP100和PVX-CP50载体GFP表达量是PVX-ori的3倍以上，而PVX-CP200、PVX-CP100和PVX-CP50载体GFP之间GFP表达无明显差异。
表2
在当前PVX植物表达载体研究基础上，本发明发现了一种能够明显提高PVX载体蛋白表达的方法。此发明涉及添加PVX病毒一段CP序列到目标基因后，在不影响其他操作的基础上，大大提高了重组蛋白的表达量，降低植物重组蛋白生产的成本。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 浙江华缔药业集团医药开发有限公司
<120> 序列的应用、重组载体及其提高PVX植物表达载体蛋白表达量的方法
<130> MP2012870
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
taccccagtt tcatagtatt ttctggtttg attgtatgaa taatataaat 50
<210> 2
<211> 100
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggctgttgtc actctaccac caccataact acgtctacat aaccgacgcc taccccagtt 60
tcatagtatt ttctggtttg attgtatgaa taatataaat 100
<210> 3
<211> 200
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cctttgtgaa gattacaaag gccagggcac aatccaacga ctttgccagc ctagatgcag 60
ctgtcactcg aggtcgtatc actggaacaa caaccgctga ggctgttgtc actctaccac 120
caccataact acgtctacat aaccgacgcc taccccagtt tcatagtatt ttctggtttg 180
attgtatgaa taatataaat 200
Claims (6)
1.CP基因的3`端至少50bP的序列在促进PVX植物表达载体中外源目的基因的表达的应用；
所述序列如SEQ ID No.1～3中任一项所示。
2.如权利要求1所述的应用，其特征在于，所述外源目的基因包括GFP基因。
3.CP基因的3`端至少50bP的序列在提高PVX植物表达载体中蛋白表达量的应用；
所述序列如SEQ ID No.1～3中任一项所示。
4.如权利要求3所述的应用，其特征在于，所述蛋白包括GFP。
5.重组载体在提高外源目的基因的表达或蛋白表达量中的应用；
所述重组载体包括PVX植物表达载体、外源目的基因以及CP基因的3`端至少50bP的序列；
所述序列如SEQ ID No.1～3中任一项所示。
6.提高PVX植物表达载体蛋白表达量的方法，其特征在于，构建重组载体，转染农杆菌，培养，提取蛋白；
所述重组载体包括PVX植物表达载体、外源目的基因以及CP基因的3`端至少50bP的序列；
所述序列如SEQ ID No.1～3中任一项所示。
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