CN108359679A - 含有人巨细胞病毒ul147基因的重组质粒、基因工程菌及其应用 - Google Patents
含有人巨细胞病毒ul147基因的重组质粒、基因工程菌及其应用 Download PDFInfo
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Abstract
本发明公开了一种含有人巨细胞病毒UL147基因的重组质粒、基因工程菌及其应用。该含有人巨细胞病毒UL147基因的重组质粒为将UL147基因插入表达型载体pET32a中获得;然后将该重组质粒转进表达菌BL21(DE3)中构建了能表达聚组氨酸标签HCMV UL147融合蛋白的工程菌株,菌株可表达获得对应的HCMV UL147融合蛋白。该融合蛋白可应用亲和层析纯化制备多克隆抗体,从而有利于探究趋化因子蛋白HCMV UL147如何规避宿主免疫系统的逃避机制,降低HCMV潜伏感染宿主细胞的数量提高机体的免疫识别。
Description
技术领域
本发明涉及生物学领域,特别涉及一种含有人巨细胞病毒UL147基因的重 组质粒、基因工程菌及其应用。
背景技术
巨细胞病毒感染是由人巨细胞病毒(human cytomegalovirus,HCMV)引起的 常见病毒性感染之一,其特征是受感染的细胞体积明显增大,细胞中出现包涵 体。在新生儿、免疫低下和免疫抑制病人(如艾滋病、器官移植或肿瘤)可引 起严重感染,甚至导致死亡。HCMV在正常人群中传播极为广泛,很少导致明 显疾病,大多数人呈潜伏感染。但这对免疫减弱或损伤的人群(免疫缺陷、器 官移植、肿瘤、艾滋病病人和孕妇)构成极大威胁。因此,亟待对于致病和免 疫机制研究,其中包括从分子水平上研究急性和潜伏感染期受染细胞的特点, 病毒对受染细胞致病效应和机体对受染细胞的免疫反应。所有病原微生物均包含和产生一种或多种独特的蛋白质,原则上即使这些分子的完整形式从未达到 胞膜,这些蛋白质可通过蛋白酶解产生小的片段,被MHC分子捕获,运送到细 胞表面,从而以多价阵列的方式被T细胞识别。这些肽可来源于感染细胞中入 侵病原微生物的特殊蛋白质,也可来源于病原体胞内复制中新生成蛋白质。识 别这些感染标志,增加感染细胞对病原体存活的抵抗、消灭感染细胞,从而消 除病原体的爆发性复制(W.E保罗,Fundamental Immunology,2003)。
趋化因子是一类可调节免疫反应的小分子家族具有细胞趋化性和细胞因子 活性。HCMV vCXCL2TB40/E在野生型毒株和临床毒株中功能域高度保守,具有 针对HLA-DQ、HLA-DP、HLA-DR高度保守的T细胞抗原表位,可以活化CTL 有助于机体清除病毒感染的细胞,防止病毒的进一步传播。Hegde等研究发现 HCMV gpUS3可在内质网中与MHC-Ⅱ类分子的α和β链结合,抑制其由内质 网向高尔基体转运,使其在细胞表面表达下降。HCMV gpUS2可与HLA-DR-α 和HLA-DM-α链结合并使其降解。HCMV gpUS2和gpUS3可以可溶性表达于细 胞外,被未感染的APC细胞摄取,抑制未感染的APC细胞呈递抗原给CD4+T 细胞。此外,MarcellaBS等研究发现人趋化因子受体类似物:HCMV基因组含 4中CC类趋化因子同源物编码基因,即UL33、UL78、US27和US28,但只有 gpUS28有趋化因子受体活性。US28类似CC类趋化因子受体,可高亲和力的结 合CC类趋化因子MCP-Ⅰ、RANES和MIP-Ⅰ。研究表明,HCMV感染可使培 养细胞的RANTES和MCP在24小时内明显上升,但此后却明显下降,而RT-PCR 证实,此时趋化因子mRNA水平并未明显下降,并进一步证实细胞表面结合的 趋化因子增加,说明CC类趋化因子被吸附在被感染细胞表面,而游离因子减少, 因而不能建立有效的趋化因子梯度,不能趋化吞噬细胞到达炎症部位,从而达 到免疫逃避的目的。
pET载体最初由Studier及其同事构建。Novagen开发的系列新的pET载体 则使目的蛋白的克隆、检测以及纯化更加容易。pET32系列载体是设计用来克 隆构建和高水平融合表达表达带有109个氨基酸Trx标签的蛋白质序列。
HCMV在与人类共进化的过程中,形成了多种分子机制逃避机体免疫系统 的监视,从而在体内长期潜伏。GpUS3是HCMV表达的一种即刻早期蛋白,可 在内质网与HLA-A,HLA-B分子的α重链结合,阻碍MHC-Ⅰ分子装配成熟及 从内质网中转运至细胞膜表面(BullockGC,et al.2001)。HCMV gpUS6在胞浆中 可与TAP结合,阻止抗原肽从胞浆转运至内质网,使MHC-Ⅰ分子不能结合抗 原肽,不能在细胞表面表达MHC-Ⅰ分子和抗原肽复合体。HCMV可编码一种 白细胞介素-10类似物cmvIL-10,cmvIL-10可降低IFN-γ的表达,导致MHC-Ⅱ 表达的分子降低。上述相关工作都为后续研究HCMV免疫逃逸提供了丰富的理 论依据(Juliet V,et al.2002)。因此,成功构建含有UL147重组质粒的基因工程 菌,并利用菌株表达获得对应的HCMV UL147融合蛋白具有重要意义。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足,提供一种含有人巨细 胞病毒UL147基因的重组质粒。
本发明的另一目的在于提供含有上述人巨细胞病毒UL147基因重组质粒的 基因工程菌。
本发明的又一目的在于提供上述含有巨细胞病毒UL147基因重组质粒的基 因工程菌的应用。
本发明的目的通过下述技术方案实现:一种含有人巨细胞病毒UL147基因 的重组质粒,含有UL147基因片段,其中,UL147基因片段的核苷酸序列如SEQ ID No:1所示。
所述的含有人巨细胞病毒UL147基因的重组质粒的构建方法,通过常规方 法将UL147基因片段插入到pET32a载体中获得。
一种含有人巨细胞病毒UL147基因重组质粒的基因工程菌,转化有上述含 有人巨细胞病毒UL147基因的重组质粒。
所述的含有人巨细胞病毒UL147基因重组质粒的基因工程菌的构建方法, 为将上述含有人巨细胞病毒UL147基因的重组质粒转化入原核表达工程菌中。
所述的基因工程菌优选为大肠杆菌;更优选为大肠杆菌BL21(DE3)。
所述的含有人巨细胞病毒UL147基因重组质粒的基因工程菌在UL147重组 蛋白制备中的应用。
一种UL147重组蛋白的制备方法,将上述含有人巨细胞病毒UL147基因重 组质粒的基因工程菌经过活化培养和发酵培养,再加入诱导剂IPTG进行诱导表 达,得到UL147重组蛋白;优选为:将上述工程菌接种至含有氨苄青霉素(Amp) 抗性的LB液体培养基中进行活化培养,然后转接到含有氨苄青霉素抗性LB液 体培养基中进行发酵培养,再加入诱导剂IPTG进行诱导表达,得到UL147重 组蛋白。
所述的诱导表达的条件优选为:在20℃诱导6h。
所述的诱导剂IPTG的诱导浓度为1mM/L。
所述的UL147重组蛋白的制备方法,还包括将获得的UL147重组蛋白进行 纯化的步骤。
所述的纯化为采用亲和层析进行纯化。
所述的含有人巨细胞病毒UL147基因重组质粒的基因工程菌在制备人巨细 胞病毒UL147趋化因子蛋白多克隆抗体中的应用;该工程菌可以表达出带有组 氨酸标签的融合蛋白。
本发明相对于现有技术具有如下的优点及效果:
1、本发明通过将UL147基因插入表达型载体pET32a中获得重组载体 pET32a-UL147,之后将其热击转进表达菌BL21(DE3)中构建了能表达聚组氨 酸标签HCMV UL147融合蛋白的工程菌株,菌株可表达获得对应的HCMV UL147融合蛋白,该融合蛋白可应用亲和层析纯化制备多克隆抗体,从而有利 于探究趋化因子蛋白HCMV UL147如何规避宿主免疫系统的逃避机制,降低 HCMV潜伏感染宿主细胞的数量提高机体的免疫识别。
2、本发明中构建含有UL147重组质粒的基因工程菌,有助于研究UL147 基因在人巨细胞病毒免疫逃避中的分子机制。一旦该分子机制被解锁,UL147 基因可能会用作人巨细胞病毒感染的治疗靶点,为人类社会做出巨大贡献。
3、在本发明pET32a-UL147重组质粒中,基因的表达是受T7噬菌体RNA 聚合酶调控的。载体的典型特点是带有pBR322的大肠菌素E1复制区,从而赋 予宿主菌氨苄青霉素抗性。编码序列在多克隆位点插入,在T7lac启动子的控 制之下,聚组氨酸标签的目的蛋白大量诱导表达,便于后续亲和纯化目的蛋白 利于制作多克隆抗体,分析免疫逃逸机制。
附图说明
图1为pET32a-UL147重组质粒构造示意图。
图2为PCR扩增产物电泳示意图;其中,泳道M为DNA marker;泳道1为UL147 PCR扩增产物。
图3为重组质粒双酶切鉴定结果图;其中,泳道M为DNA marker;泳道1为 重组质粒pET32a-UL147经BamHⅠ、HindⅢ双酶切后结果(与预期结果大小一 致)。
图4为重组融合蛋白UL147(未纯化)的SDS-PAGE结果示意图;其中,泳道M 为蛋白质Marker;箭头所示为重组融合蛋白UL147,其为35KD。
图5为重组蛋白的Western blot结果图;其中,泳道M为预染蛋白质Marker; 箭头所示为重组融合蛋白UL147,其为35KD。
图6为重组融合蛋白UL147纯化的SDS-PAGE结果示意图;其中,泳道M为蛋 白质Marker;箭头所示为重组融合蛋白UL147,其为35KD。
图7为重组蛋白纯化的Western blot结果图;其中,泳道M为预染蛋白质 Marker;箭头所示为重组融合蛋白UL147,其为35KD。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于 此。
一、质粒构建
(1)UL147基因引物设计
根据GennBank上公布的TB40/E(KF297339.1)病毒株基因组中UL147基 因的序列全长480bp(核苷酸序列如SEQ ID No:1所示,蛋白翻译产物如SEQ ID No:2所示),以该序列为模板,以pET32a载体(优宝生物有限公司)为克 隆载体和表达载体,使用DNAstar软件,选取合适的酶切位点进行引物设计。 设计好的引物如下表:
表1 UL147引物设计表
注:F(上游引物);R(下游引物)。
(2)UL147基因PCR
根据已发表在GenBank上的HCMV TB40/E-BAC的基因序列(GenBank: EF999921.1,引用文章(Cloning and sequencing of a highly productice, endotheliotropicvirus strain derived from human cytomegalovirus TB40/E))为模 板,在以下反应体系中进行PCR:
表2 PCR反应体系表
试剂名称 | 体积(20μl) |
无菌ddH2O | 14.9μl |
5×PCR buffer(with Mg2+) | 5μl |
dNTP | 2μl |
模板 | 1μl |
上游引物 | 1μl |
下游引物 | 1μl |
Fast pfu酶 | 0.1μl |
PCR扩增条件见下表:
表3 PCR反应条件设置表
PCR产物经1%的琼脂糖凝胶分析,并通过胶回收试剂盒回收PCR产物。
PCR产物回收步骤如下:
①PCR产物经电泳后,在紫外条件下切取含目的条带的凝胶置于干净的1.5 mL的EP管中;
②在EP管中加入与凝胶等体积的XP2溶液;
③将混有XP2溶液的凝胶块的1.5mL的EP管置于水浴锅中,57℃加热, 每隔2~3min颠倒混匀,直至凝胶块完全融化即可;
④待完全融化后的凝胶冷却,便可将其斡旋混匀并短暂离心,最后将其加 入事先组装好的硅胶柱,室温,10000×g,离心1min,倾倒收集管中的废液;
⑤再加入300μL XP2溶液到硅胶柱中,室温10000×g离心1min后,将收 集管中的液体倒掉;
⑥用700μL SPW洗涤溶液洗涤硅胶柱,室温10000×g离心1min,将收集 管中的液体倒掉;
⑦重复6步骤一次;
⑧空甩硅胶柱,室温12000×g离心2min,去除多余的SPW洗涤溶液;
⑨取出硅胶柱并将其放入到灭菌的1.5mL EP管中,室温放置2min后,向 硅胶柱中加入30μL事先已加热到65℃无菌ddH20,室温静置1min后,12000×g 离心1min,取出已废弃的硅胶柱,EP管中收集的液体即为目的DNA溶液;
⑩检测回收的目的DNA浓度及纯度,样品放于-20℃保存。
实验结果:PCR扩增产物如图2所示,与预期大小结果一致。
(3)产物纯化试剂盒回收PCR产物
①将PCR扩增后产物转移到一个干净的1.5mL EP管,根据PCR扩增体系 加入4~5倍的结合液CP,充分混匀;
②将上一步所得溶液加入吸附柱EC中(吸附柱放入收集管中),室温放置 1min,10000×g,离心1min,倾倒收集管中的废液;
③加入700μl漂洗液WB(先检查是否已加入无水乙醇),10000g离心1min, 弃掉废液;
④重复3步骤一次;
⑤将吸附柱EC放回空收集管中,10000×g,离心1min,尽可能去除漂洗液, 漂洗液中残留乙醇会抑制下游反应;
⑥取出吸附柱EC,放入一个干净的离心管中,在吸附膜的中间部位加30~ 50μl洗脱缓冲液EB(洗脱缓冲液事先在65~70℃水浴中加热效果更好),室温 放置2min,13000×g离心1min。如果需要较多量DNA,可将得到的溶液重新 加入吸附柱中,离心1min。取出已废弃的硅胶柱,EP管中收集的液体即为目 的DNA溶液;
⑦检测回收的目的DNA浓度及纯度,以更好的用于后续实验。样品放于 -20℃保存。
(4)pET32a质粒抽提
①取出过夜培养好的含有pET32a质粒的DH5α菌液,并将菌体加到1.5mL EP管中,12000×g室温离心1min,弃上清,重复步骤,将5mL菌液全部收集 到1.5mL EP管中;
②吸净多余的上清后,加250μl solution I到EP管中,用移液器轻轻吹打 菌体沉淀,待菌体重悬后,放于涡旋振器上,使其完全混匀;
③再加入250μl solution II,轻轻地上下颠倒混匀数次,颠倒时间要把握好, 控制在2min~5min之间,颠倒完之后,液体一般会透明澄清;
④最后加入350μl solution III,轻轻地上下颠倒混匀数次,至有白色沉淀形成,颠倒混匀次数不能过多,紧接着放于小型离心机中,室温,13000×g离心 10min;
⑤离心结束后,将移液枪把上清液转移到已标记好的硅胶柱中,硅胶柱放 进2ml的离心管里,勿将白色沉淀物吸入硅胶柱中,放于小型离心机中,室温 10000×g,离心1min;
⑥倒掉收集管中的废液,吸取500μl HB溶液,加入到硅胶柱中,室温 10000×g离心1min;
⑦倒掉收集管中的废液,加700μl DNA Wash Buffer到硅胶柱中,(先检查 DNAWash Buffer是否已加入无水乙醇)室温10000×g离心1min;重复8步骤 一次;
⑧室温13000×g离心2min,去除多余的DNA Wash Buffer;
⑨取出硅胶柱将其放入到灭菌的1.5mL EP管中,向硅胶柱中加入30μl~ 50μl事先已加热到65℃无菌ddH20,室温静置1~2min后,12000×g离心1min, 于-20℃保存。
(5)载体和目的基因的酶切及回收
①pET32a载体和UL147基因PCR产物双酶切体系如下表:
表4 PCR产物双酶切体系表
组分 | pET32a质粒DNA(μL) | PCR产物(μL) |
10×FastDigest Buffer | 2 | 2 |
DNA | 16(1μg) | 6.25(0.2μg) |
FastDigest enzyme Sal I | 1 | 1 |
FastDigest enzyme BamH I | 1 | 1 |
ddH2O | 0 | 19.75 |
总体积 | 20 | 30 |
将抽提好的质粒与回收的PCR产物于37℃恒温水浴14min进行双酶切
②pET32a双酶切后需要胶回收,方法同步骤(2)。
③UL147目的基因双酶切回收
Ⅰ根据双酶切体系加入4~5倍的结合液CP,充分混匀;
Ⅱ、将上一步所得溶液加入吸附柱EC中,室温放置1min,10000×g,离心 1min,倒掉收集管中的废液;
Ⅲ、加入700μl漂洗液WB(先检查是否已加入无水乙醇),10000×g,离 心1min,弃掉废液;
Ⅳ、重复3步骤一次;
Ⅴ、将吸附柱EC放回收集管中,10000×g,离心1min,尽可能除去漂洗液, 以免残留乙醇抑制下游反应;
Ⅵ、取出吸附柱EC,放入一个干净的离心管中,在吸附膜的中间部位加30~ 50μl洗脱缓冲液EB(洗脱缓冲液事先在65~70℃水浴中加热效果更好);室温 放置2min,13000×g离心1min。如果需要较多量DNA,可将得到的溶液重新 加入吸附柱中,离心1min。取出已废弃的硅胶柱,EP管中收集的液体即为目 的DNA溶液;
Ⅶ、检测回收的目的DNA浓度及纯度,以更好的用于后续实验。样品放于 -20℃保存。
(6)UL147双酶切回收产物与pET32a质粒双酶切回收产物连接反应
连接反应体系如下表:
表5连接反应体系表
成分 | 体积(总20μl) |
pET32a质粒 | 5μl |
gL(UL147) | 10μl |
10×T4 Ligation Buffer | 2μl |
T4 DNA Ligase | 1μl |
ddH2O | 2μl |
连接反应条件为置于16℃连接仪中连接16h以上;获得重组质粒 pET32a-UL147。
(7)大肠杆菌BL21(DE3)感受态细胞的制备
①从-80℃冰箱取出大肠杆菌BL21(DE3),于无抗性的LB固体培养基上 划线接种,37℃培养12h;
②在划线过夜后的LB平板上,挑取单菌落,接种于4mL LB液体培养基(含 Amp,终浓度为100μg/mL)中37℃培养过夜;
③将培养得到产物次日按1%(v/v)的比例,接种于500mL LB液体培养基 中,37℃培养;
④当培养物OD600达0.5~0.6时,将培养物置冰上10~15min,8000r/min 离心10min收集菌体;
⑤用预冷的TB buffer轻轻地将沉淀悬浮,8000r/min离心30min,如此重复 洗涤2次;
⑥用18.6ml预冷TB buffer和1.4ml DMSO(二甲基亚砜)的轻轻将沉淀悬 浮,-80℃保存备用。
(8)重组质粒转化进大肠杆菌BL21(DE3)
①取重组质粒pET32a-UL147 1μl加入BL21(DE3)感受态细胞中,冰上静 置30min;
②将混有质粒的BL21(DE3)感受态细胞放入恒温水浴锅中42℃90S热击;
③热击后立即冰浴3min;
④冰浴后加入800μl SOB液体培养基(SOB培养基配方:2%(w/v)Tryptone、 0.5%(w/v)Yeast Extract、0.05%(w/v)氯化钠、2.5mM氯化钾、10mM氯化 镁),37℃培养1h,取出后将培养物8000r/min离心5min,弃去上清,只留下约 100μl菌液,吹打混匀后将其涂于LB平板(含Amp),放置37℃培养箱中倒置过 夜培养12~24h即可看到单菌落。
(9)重组质粒鉴定
①无菌操作下,挑取单菌落,接种于10mL LB液体培养基(含Amp)中,37℃ 培养8h后提取质粒,把1%琼脂糖核酸凝胶电泳鉴定为阳性的pET32a-UL147 重组质粒作为模板,进行PCR鉴定。
表6 PCR反应体系表
试剂名称 | 体积(20μl) |
无菌ddH2O | 14.9μl |
5×PCR buffer(with Mg2+) | 5μl |
dNTP | 2μl |
模板 | 1μl |
上游引物 | 1μl |
下游引物 | 1μl |
Fast pfu酶 | 0.1μl |
PCR扩增条件见下表:
表7 PCR反应条件设置表
PCR扩增结束后,反应产物跑1%的琼脂糖凝胶电泳,凝胶成像仪拍照,并 分析、保存实验结果。
实验结果:组质粒双酶切鉴定结果如图3所示,重组质粒pET32a-UL147 经BamHⅠ、HindⅢ双酶切后结果与预期结果大小一致。
②双酶切鉴定
Ⅰ.抽提重组质粒pET32a-UL147同步骤(4);
Ⅱ.重组质粒pET32a-UL147双酶切鉴定同步骤(5)。
(10)质粒样品测序
将可以双酶切出目的基因大小片段的质粒样品送公司(南京锐真生物技术 有限公司)测序,测序结果出来后,将测序结果与GennBank下载的UL147 (vCXCL2)基因序列使用BLAST软件进行比对,分析比对结果。测序结果与 下载序列匹配,可以进行下一步实验。
二、UL147重组蛋白的原核表达
(1)UL147重组蛋白的诱导表达鉴定
①重组载体pET32a-UL147诱导表达条件优化:pET32a-UL147表达趋化因 子vCXCL2最佳诱导条件是:20℃、1%(v/v)浓度为1mM的IPTG、6h。
②重组蛋白UL147的鉴定
Ⅰ.把鉴定好的pET32a-UL147重组BL21(DE3)菌按1%接种于4mL LB 液体培养基(含Amp)中,37℃静止培养过夜。次日按1%将活化的菌液接种 于4mL LB液体培养基(含Amp)中,37℃当菌液OD600约为0.5时加入1% (v/v)浓度为1mM的IPTG,20℃培养6h。8000r/min离心1min收集菌体,加 入4×上样缓冲液(含DTT),用漩涡震荡器混匀,沸水浴中煮沸10min,l0000r/min 离心30s,取15μl上清,以12%分离胶5mL,8%浓缩胶2mL进行配胶进行SDS 聚丙烯酞胺凝胶电泳(SDS-PAGE)。
表8 12%分离胶配制表
混匀后将试剂灌入玻璃板间,以水封顶,注意使液面水平,凝胶完全聚合 需要30~60min;
配置5%浓缩胶见下表:
表9 5%浓缩胶配制表
试剂名称 | 体积(总5ml) |
无菌ddH2O | 3.4ml |
30%丙烯酰胺贮存胶 | 830μl |
浓缩胶缓冲液 | 630μl |
10%SDS | 50μl |
10%AP | 85μl |
TEMED | 5μl |
将分离胶上的水倒去,并用滤纸吸干多余水份,加入上述混合液,立即将 梳子插入玻璃板间,完全聚合需15~30min;
Ⅱ.Western Blot步骤如下:
①首先将细胞蛋白经12%SDS-PAGE凝胶电泳进行分离并转移至PVDF膜 上;
②用5%的脱脂奶粉室温封闭1h;
③封闭完的PVDF膜与anti-His鼠抗(1:1000)4℃共孵育过夜;
④用含有1/2000吐温的TBST洗3次,每次20min;
⑤洗后的膜与荧光二抗山羊抗鼠(1:5000)4℃共孵育2~3h;
⑥再用含有1/2000吐温的TBST洗3次,每次20min;
⑦最后通过LI-COR ODYSSEY FC成像系统进行曝光进行免疫印迹分析。
实验结果:重组融合蛋白UL147(未纯化)的SDS-PAGE结果如图4所示; 重组蛋白的Western blot结果如图5所示。
三、UL147重组蛋白的纯化
(1)超声波破菌
①加入1L(1/20细胞生长体积)的1×PBS缓冲液重悬菌体,4℃,6,500×g, 离心10min尽可能弃尽上清;
②加入的50ml 1×PBS缓冲液重悬菌体,采用超声波破碎仪进行破菌,破菌 参数设置为:150W,工作15s,停5s,破碎(30min~1h)。
③破菌完成后,10000×g,4℃离心15min以上,收集上清液体1.5ml,用 移液枪吸100μl沉淀,加到1.5ml EP管内,跑SDS-PAGE蛋白胶分析样品;
④剩下的沉淀加入30ml的平衡缓冲液,37℃震荡1h,冰上0.22μm滤膜过 滤备用。
(2)UL147重组蛋白亲和层析纯化
①10倍柱体积的ddH2O(0.22μm)清洗Ni层析柱;
②10倍柱体积的平衡缓冲液(0.22μm)清洗Ni层析柱;
③平衡泵泵入UL147重组蛋白溶液;
④分别用5倍柱体积5mM咪唑、50mM咪唑、100mM咪唑、150mM咪唑、 200mM咪唑、500mM咪唑洗脱。
(3)UL147纯化重组蛋白的鉴定
UL147重组蛋白的亲和层析纯化鉴定同步骤二(1)②。
实验结果:重组融合蛋白UL147纯化的SDS-PAGE结果如图6所示;重组 融合蛋白纯化的Western blot结果如图7所示。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施 例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替 代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 暨南大学
<120> 含有人巨细胞病毒UL147基因的重组质粒、基因工程菌及其应用
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 480
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> UL147基因
<400> 1
tcaccagcgc agtctgaagt ggtggtagtt caatttcttg gcgtatttcc agagaaaggc 60
tttgtaggcc gtaggggctg gccaggcacc aaactcaata ttggtagaca ctacgtcgta 120
aatgcgttgt tcttcgtcta agattaaccg aaaaaatagc cggttgatgt gacgacgcac 180
agcttgcgcg ttaggattga gacacttggt gcccttgtcc tttaaaatag ccagcacttc 240
ctgacgattg cagctttcgc tcgctgcgat tggcttaagc agttgagttc cgactggcag 300
ggtattcaac agaatttggt tgttgcaacg acagcgcttg tcgtaatctt ccaattctaa 360
gagatagacg aataggggac acgtggaaaa taacacatat gcggtcaaat acaggtatcg 420
taccgataag actttgatgt gcgaatttgg aatcggatgg tgtaaccatg tcaaaaccat 480
<210> 2
<211> 159
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> UL147蛋白
<400> 2
Met Val Leu Thr Trp Leu His His Pro Ile Pro Asn Ser His Ile Lys
1 5 10 15
Val Leu Ser Val Arg Tyr Leu Tyr Leu Thr Ala Tyr Val Leu Phe Ser
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Thr Cys Pro Leu Phe Val Tyr Leu Leu Glu Leu Glu Asp Tyr Asp Lys
35 40 45
Arg Cys Arg Cys Asn Asn Gln Ile Leu Leu Asn Thr Leu Pro Val Gly
50 55 60
Thr Gln Leu Leu Lys Pro Ile Ala Ala Ser Glu Ser Cys Asn Arg Gln
65 70 75 80
Glu Val Leu Ala Ile Leu Lys Asp Lys Gly Thr Lys Cys Leu Asn Pro
85 90 95
Asn Ala Gln Ala Val Arg Arg His Ile Asn Arg Leu Phe Phe Arg Leu
100 105 110
Ile Leu Asp Glu Glu Gln Arg Ile Tyr Asp Val Val Ser Thr Asn Ile
115 120 125
Glu Phe Gly Ala Trp Pro Ala Pro Thr Ala Tyr Lys Ala Phe Leu Trp
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Lys Tyr Ala Lys Lys Leu Asn Tyr His His Phe Arg Leu Arg Trp
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<210> 3
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> UL147引物-F
<400> 3
cgcggatcca tggttttgac atggttacac catc 34
<210> 4
<211> 36
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> UL147引物-R
<400> 4
gcgcggaagc ttctatcacc agcgcagtct gaagtg 36
Claims (8)
1.一种含有人巨细胞病毒UL147基因的重组质粒,其特征在于:含有UL147基因片段,其中,UL147基因片段的核苷酸序列如SEQ ID No:1所示。
2.一种含有人巨细胞病毒UL147基因重组质粒的基因工程菌,其特征在于:转化有权利要求1中所述的含有人巨细胞病毒UL147基因的重组质粒。
3.根据权利要求2所述的含有人巨细胞病毒UL147基因重组质粒的基因工程菌,其特征在于:所述的基因工程菌为大肠杆菌。
4.权利要求2或3所述的含有人巨细胞病毒UL147基因重组质粒的基因工程菌在UL147重组蛋白制备中的应用。
5.一种UL147重组蛋白的制备方法,其特征在于:将权利要求2或3中所述的含有人巨细胞病毒UL147基因重组质粒的基因工程菌经过活化培养和发酵培养,再加入诱导剂IPTG进行诱导表达,得到UL147重组蛋白。
6.根据权利要求5所述的UL147重组蛋白的制备方法,其特征在于:
所述的诱导表达的条件为:20℃诱导6h;
所述的诱导剂IPTG的诱导浓度为1mM/L。
7.根据权利要求5所述的UL147重组蛋白的制备方法,其特征在于:还包括将获得的UL147重组蛋白进行纯化的步骤;
所述的纯化为采用亲和层析进行纯化。
8.权利要求2或3所述的含有人巨细胞病毒UL147基因重组质粒的基因工程菌在制备人巨细胞病毒UL147趋化因子蛋白多克隆抗体中的应用。
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