CN113845597A - CTB-Cap融合蛋白及其编码基因与应用 - Google Patents
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Abstract
本发明属于生物技术制药工业中基因工程领域,具体是一种CTB‑Cap融合蛋白及其编码基因与应用。CTB‑Cap融合蛋白的氨基酸序列如SEQ ID NO:2所示。CTB‑Cap融合蛋白的编码基因的碱基序列如SEQ ID NO:1所示。本发明所述的CTB‑Cap融合蛋白利用了乳酸乳球菌安全无毒、易于培养、外源蛋白表达高效、表达的产物无需纯化等优点,以乳酸乳球菌表达CTB‑Cap融合蛋白,将此为猪圆环病毒疫苗的研制提供了一定的理论依据。
Description
技术领域
本发明属于生物技术制药工业中基因工程领域,具体是一种CTB-Cap融合蛋白及其编码基因与应用。
背景技术
目前,防治猪圆环病毒2型(Porcine circovirus 2,PCV2)最有效的方式是疫苗,PCV2可以通过黏膜传染,故黏膜防护对阻断其传播有着重要的作用。目前已上市的PCV2亚单位疫苗国外有勃林格公司研制的英特威公司研制的以及先灵葆雅公司研制的PCV,国内的有武汉中博、扬州优邦、青岛易邦和普莱柯生物的亚单位疫苗。商业化PCV2疫苗以灭活苗和亚单位疫苗为主。但灭活苗难制备,病毒滴度低,生产周期长,生产成本高,不能完全阻断PCV2的传播。亚单位疫苗多使用大肠杆菌表达载体或杆状病毒表达载体;大肠杆菌虽易于培养,但容易形成包涵体增加纯化成本;而杆状病毒表达载体表达操作难,生产成本大。这些PCV2疫苗诱导的免疫反应主要为细胞及体液免疫,忽略了黏膜免疫。所以能够刺激机体产生黏膜免疫的疫苗在防控PCV2方面有巨大潜力。乳酸菌作为全世界公认的食品安全级微生物,蛋白产量高,培养周期短,生产成本低,免疫原性低,并能够将抗原携带至肠黏膜处,是很好的能够刺激黏膜免疫的活载体。但经黏膜接种的疫苗一般免疫应答弱,抗体维持时间较短,免疫效果不理想,有效的黏膜免疫增强剂的使用就显得尤为重要。
本发明以“免疫原性+免疫增强”的理念,将PCV2基因和霍乱毒素B亚单位(Choleratoxin B subunit,CTB)基因进行串联,利用基因工程技术构建能够表达CTB-Cap融合蛋白的重组乳酸乳球菌,通过动物实验验证重组乳酸乳球菌的免疫原性,以期为PCV2重组活载体疫苗研究奠定一定的理论基础。
发明内容
本发明利用CTB-Cap和乳酸乳球菌的优点,提供了一种CTB-Cap融合蛋白及其编码基因与应用。
本发明是通过以下技术方案实现的:CTB-Cap融合蛋白,其氨基酸序列如SEQ IDNO:2所示。
本发明进一步提供了所述CTB-Cap融合蛋白在制备猪圆环病毒2型疫苗中的应用。
本发明进一步提供了所述CTB-Cap融合蛋白的制备方法,包括以下步骤:
表达载体的构建:全基因组合成含有CTB-Cap和乳酸菌表达载体pNZ8148的重组质粒pNZ8148-CTB-Cap,通过双酶切和PCR检测,筛选出含有CTB-Cap基因序列的克隆;
乳酸乳球菌表达系统的构建:通过电转化的方法,将获得的重组质粒pNZ8148-CTB-Cap转入乳酸菌感受态NZ9000;将转化后的菌体悬液涂布于含有氯霉素的GM17平板上,30℃培养;挑取单克隆,使用不同浓度的Nisin诱导表达CTB-Cap融合蛋白,超声破碎菌体;SDS-PAGE和Western blot检测确认阳性克隆。
本发明还提供了CTB-Cap融合蛋白的编码基因,其碱基序列如SEQ ID NO:1所示。
在本发明中,所述CTB-Cap融合蛋白的编码基因是由编码CTB的cDNA序列、猪圆环病毒2型Cap蛋白的cDNA序列、6个组氨酸cDNA序列和限制性内切酶Nco I和Hind III位点的cDNA序列以及符合乳酸乳球菌表达偏好性的基因序列组成的。
本发明所述的CTB-Cap融合蛋白利用了乳酸乳球菌安全无毒、易于培养、外源蛋白表达高效、表达的产物无需纯化等优点,以乳酸乳球菌表达CTB-Cap融合蛋白,将此为猪圆环病毒疫苗的研制提供了一定的理论依据。
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,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为重组质粒pNZ8148-CTB-Cap鉴定结果图。两图中A:阳性质粒酶切鉴定结果,B:阳性菌落PCR鉴定结果。图中1:Marker,2:CTB-Cap质粒,3:双酶切后的质粒,4:阳性CTB-Cap菌落。
图2为CTB-Cap融合蛋白鉴定结果图。其中A:SDS-PAGE分析,B:Western blot分析(抗CTB),C:Western blot分析(抗his)。图中M:蛋白分子质量标准,1:未诱导的重组乳酸菌,2:30ng/ml诱导4h破碎上清,3:50ng/ml诱导4h破碎上清,4:100ng/ml诱导4h破碎上清,5:30ng/ml诱导4h破碎沉淀,6:50ng/ml诱导4h破碎沉淀,7:100ng/ml诱导4h破碎沉淀。
图3蛋白浓度测定标准曲线。
图4各处理组体重变化。
图5 CTB-Cap融合蛋白免疫小鼠后血清中特异性抗体水平。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
1、所用试剂及其配制
(1)LB液体培养基:1g蛋白胨、0.5g酵母粉和1g NaCl融于100mL dH2O中,高压灭菌;
(2)GM17液体培养基:4.72g GM17融于100mL dH2O,高压灭菌;
(3)GM17固体培养基:4.72g GM17和1.5g琼脂粉融于100mL dH2O中,高压灭菌;
(4)GMMC恢复培养基:4.72g/100mL GM17、20mM MgCl2和2mM CaCl2混匀;
(5)电泳缓冲液:3.03g Tris、18.77g甘氨酸和1g SDS融于1000mL dH2O中;
(6)转膜缓冲液:5.8g Tris、2.9g甘氨酸、0.37g SDS和200mL甲醇融于1000mLdH2O中;
(7)TBST:4.4g NaCl、5mL Tris-HCl(PH=7.5)和250μL Tween-20融于500mLdH2O。
2、重组质粒pNZ8148-CTB-Cap获得及鉴定
寻找NCBI登陆的CTB(JF274975.1)和PCV2 Cap(DQ235696.1)以及表达载体pNZ8148的基因序列,进行优化后,送至钟鼎生物进行全基因组合成,将获得的重组质粒pNZ8148-CTB-Cap转入MC1061克隆菌株,挑取阳性克隆子扩大培养,提取重组质粒。将重组质粒用Nco I和Hind III 37℃3h进行双酶切鉴定;重组质粒使用引物(F:5'ACGCGAGCATAATAAACGG 3'、R:5'CGAAAGCGAAATCAAACGA 3')进行PCR检测。由图1-A可知,表明重组质粒pNZ8148-CTB-Cap构建成功。
3、表达载体的构建
将1μL鉴定正确的重组质粒pNZ8148-CTB-Cap和40μL乳酸菌感受态NZ9000轻柔均匀混合后,加到冰浴的0.2cm电转杯中;设定电击参数为2000V,25μF,200Ω,将电转杯置于电击槽内,电击;电击结束后立即加入1mL GMMC恢复培养基,冰浴5min,将电转杯内全部液体转入1.5mL EP管中,30℃培养1h;分别取10μL、100μL、900μL于含有10μg/mL氯霉素的GM17固体培养板上,待菌液完全吸收后,30℃培养,直至转化子出现。挑取转化子使用引物(F:5'ACGCGAGCATAATAAACGG 3'、R:5'CGAAAGCGAAATCAAACGA 3')进行PCR检测。由图1-B可知pNZ8148-CTB-Cap被转入到乳酸乳球菌中,重组乳酸乳球菌构建成功。
4、CTB-Cap融合蛋白的表达
挑取鉴定正确的转化子接种于5mL含有10μg/mL氯霉素的GM17液体培养基中,30℃225rpm过夜培养;将上述培养液接种于100mL含有10μg/mL氯霉素的GM17液体培养基中,30℃225rpm培养至菌体OD600约为0.4;取1mL培养液,10000rpm室温离心2min,弃上清,用1×上样buffer重悬菌体沉淀;向剩余的培养液中分别加入Nisin至终浓度为30、50、100ng/mL,30℃培养4h,诱导CTB-Cap融合蛋白表达,筛选最佳诱导浓度;诱导结束后,取出1mL培养液,10000rpm室温离心2min,沉淀用PBS清洗2次后,重悬;将重悬菌液超声波破碎;超声后,分别取上清液与沉淀液加入上样buffer液重悬。将上述获得的所有样品进行SDS-PAGE和Western Blot检测。由图2可知,重组乳酸乳球菌经Nisin诱导后成功表达CTB-Cap融合蛋白,30ng/mL、50ng/mL和100ng/mL的Nisin均能诱导重组乳酸乳球菌表达融合蛋白。在统一上样量的基础上,Nisin浓度在50ng/mL和100ng/mL诱导表达的蛋白量没有差别,且比30ng/mL nisin诱导表达的蛋白多,故选择用50ng/mL Nisin为最佳诱导浓度。
5、CTB-Cap融合蛋白产量的测定
将浓度为0.023mg/mL重组Cap蛋白按下表进行稀释:
诱导100mL重组乳酸乳球菌表达CTB-Cap融合蛋白,表达结束后,10000rpm室温离心2min收集菌体,沉淀用PBS清洗2次,用1mL PBS重悬沉淀后超声破碎,得到含有CTB-Cap融合蛋白的悬浊液,用PBS按1:10000进行稀释。
将鼠源抗Cap蛋白单克隆抗体用蛋白包被液按1:500稀释后,包被ELISA板,每孔100μL,4℃过夜;弃掉包被液,每孔用300μL PBST清洗2次,每次5min;每孔加入200μL 5%脱脂奶粉,37℃封闭1.5h;弃掉封闭液,每孔分别加入100μL含有CTB-Cap融合蛋白的稀释液和按上表稀释的重组Cap蛋白,每孔一个重复,37℃孵育1h;弃掉蛋白稀释液,每孔用300μLPBST清洗4次,每次5min;每孔加入100μL用封闭液1:10000稀释HRP标记的圆环抗体,37℃孵育30min;弃掉圆环抗体,每孔用300μL PBST清洗4次,每次5min;每孔加入100μL TMB显色液,37℃避光孵育10min;每孔加入50μL ELISA终止液终止反应,酶标仪检测OD450吸光值。由图3展示的蛋白浓度测定标准曲线和酶标仪测定含有CTB-Cap融合蛋白的悬浊液在450nm处的OD值为1.54925,可以计算出CTB-Cap融合蛋白表达量约为50.5mg/100mL。
6、小鼠免疫保护试验
6.1试验分组及处理
阳性疫苗试验组:猪圆环病毒2型基因工亚单位疫苗(大肠杆菌源)50μL/只;
2.5×1010试验组:25μL(1010CFU)重组乳酸乳球菌;
5×109试验组:25μL(109CFU)重组乳酸乳球菌;
5×108试验组:25μL(108CFU)重组乳酸乳球菌;
PBS试验组:25μL PBS。
取25只6周雌性BALB/C小鼠,随机分成5组,每组5只。按上述分组,阳性疫苗试验组小鼠经腿部肌肉注射阳性疫苗50μL,2.5×1010试验组、5×109试验组和5×108试验组经分别经背部皮下注射免疫25μL 2.5×1010CFU、5×109CFU和5×108CFU重组乳酸乳球菌,PBS试验组经背部皮下注射25μL PBS作为对照。除阳性疫苗组免疫一次外,其它组均免疫2次,间隔两周。
6.2重组乳酸乳球菌对小鼠增重的影响
试验期间,每周记录小鼠的体重。由图4可知,各组之间体重均无显著差异(p>0.05)。
6.3小鼠血清中抗PCV2 Cap特异性IgG的检测
首免后14、21、28、35、42d,从小鼠眼眶后静脉丛采血,分离血清,用1%BSA-PBST封闭液稀释血清,测定PCV2特异性抗体水平。
重组Cap蛋白用包被液稀释至0.1mg/mL后,每孔100μL,包被ELISA板,4℃过夜;弃掉包被液,每孔用300μL PBST清洗3次,每次5min;每孔加入200μL 1%BSA-PBST,37℃封闭3h;弃掉封闭液,每孔用300μL PBST清洗3次,每次5min;每孔加入100μL稀释后的血清,每孔一个重复,37℃孵育1h;弃掉血清,每孔用300μL PBST清洗3次,每次5min;每孔加入100μL封闭液1:10000稀释羊抗鼠HRP-IgG二抗,37℃孵育30min;弃掉二抗,每孔用300μL PBST清洗3次,每次5min;每孔加入100μL TMB显色液,37℃避光孵育10min;每孔加入50μL ELISA终止液终止反应;酶标仪检测OD450吸光值。由图5可知,首免后21d和28d,2.5×1010试验组比较各试验组有升高的趋势,但与5×109试验组、5×108试验组和PBS组相比显著差异(p>0.05)。首免后35d和42d,与PBS组相比,2.5×1010试验组显著升高(p<0.05),5×109试验组、5×108试验组显著差异(p>0.05);表明构建的重组乳酸乳球菌经皮下免疫具有免疫原性。
7、数据分析
数据均经GraphPad PrismTM 5(GraphPad Software,Inc.California,USA)数据分析软件中One-way ANOVA确定各组数据间的是否存在差异性,结果均以Mean±standarderrors mean(SEM)表示。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
<110>山西农业大学
<120>CTB-Cap融合蛋白及其编码基因与应用
<160>2
<210>1
<211>1289
<212>DNA
<213>人工序列
<220>
<223>CTB-Cap融合蛋白的编码基因
<400>1
AGGAACTACA AAATAAATTA TAAGGAGGCA CTCACCATGG GCAAAAAAAA GATTATCTCA 60
GCTATTTTAA TGTCTACAGT GATACTTTCT GCTGCAGCCC CGTTGTCAGG TGTTTACGCT 120
ACCTATCCAA GAAGAAGATA TCGTAGACGT CGTCATCGTC CAAGATCACA TCTTGGTCAA 180
ATTTTACGTC GTCGTCCTTG GTTATTACAT CCAAGACATA GATATCGTTG GCGTAGAAAA 240
AATGGTATTT TTAATACCCG TCTTAGCCGT ACATTTGGAT ATACTATTAA GCGTACCACC 300
GTTAAAACCC CTTCTTGGGC TGTTGATATG ATGAGATTCA ATATTAATGA TTTTCTTCCA 360
CCAGGTGGTG GATCTAATCC AAGATCTGTT CCATTTGAAT ATTATCGTAT TCGTAAAGTC 420
AAAGTCGAAT TTTGGCCATG TTCACCTATT ACACAAGGTG ACCGTGGAGT TGGTTCATCA 480
GCTGTTATTT TAGATGATAA TTTTGTCACC AAAGCTACCG CATTAACTTA TGATCCATAT 540
GTTAATTATA GCAGCCGTCA TACTATTACC CAACCTTTTT CATATCACAG CAGATATTTT 600
ACCCCAAAAC CAGTTCTTGA TAGCACTATT GATTATTTTC AACCAAATAA TAAGCGTAAT 660
CAACTTTGGC TTCGTCTTCA AACTGCTGGA AATGTTGATC ATGTTGGATT AGGTACTGCA 720
TTTGAAAATA GCATATATGA TCAAGAATAT AATATTCGTG TCACCATGTA TGTCCAATTT 780
CGTGAATTCA ATCTTAAAGA TCCACCACTT AATCCAGGTG GAGGTGGTTC TGGTGGAGGT 840
GGATCAGGAG GTGGTGGTTC AACTCCTCAA AATATTACAG ATCTTTGTGC CGAATATCAC 900
AATACACAAA TATATACCCT TAATGATAAA ATTTTTAGCT ATACCGAAAG CCTTGCCGGT 960
AAAAGAGAAA TGGCAATTAT TACTTTTAAA AATGGTGCTA TTTTTCAAGT CGAAGTCCCA 1020
GGTTCTCAAC ATATTGATTC ACAAAAGAAA GCTATTGAAC GTATGAAAGA TACCCTTCGT 1080
ATTGCTTATC TTACCGAAGC TAAAGTTGAA AAACTTTGTG TCTGGAATAA TAAGACCCCA 1140
CATGCAATTG CTGCTATTTC AATGGCAAAT CATCACCATC ATCATCATTG ATCTAGAGAG 1200
CTCAAGCTTT CTTTGAACCA AAATTAGAAA ACCAAGGCTT GAAACGTTCA ATTGAAATGG 1260
CAATTAAACA AATTACAGCA CGTGTTGCT 1289
<210>2
<211>384
<212>PRT
<213>人工序列
<220>
<223>CTB-Cap融合蛋白
<400>2
MGKKKIISAI LMSTVILSAA APLSGVYATY PRRRYRRRRH RPRSHLGQIL RRRPWLLHPR 60
HRYRWRRKNG IFNTRLSRTF GYTIKRTTVK TPSWAVDMMR FNINDFLPPG GGSNPRSVPF 120
EYYRIRKVKV EFWPCSPITQ GDRGVGSSAV ILDDNFVTKA TALTYDPYVN YSSRHTITQP 180
FSYHSRYFTP KPVLDSTIDY FQPNNKRNQL WLRLQTAGNV DHVGLGTAFE NSIYDQEYNI 240
RVTMYVQFRE FNLKDPPLNP GGGGSGGGGS GGGGSTPQNI TDLCAEYHNT QIYTLNDKIF 300
SYTESLAGKR EMAIITFKNG AIFQVEVPGS QHIDSQKKAI ERMKDTLRIA YLTEAKVEKL 360
CVWNNKTPHA IAAISMANHH HHHH 384
Claims (4)
1.CTB-Cap融合蛋白,其氨基酸序列如SEQ ID NO:2所示。
2.权利要求1所述CTB-Cap融合蛋白在制备猪圆环病毒2型疫苗中的应用。
3.权利要求1所述CTB-Cap融合蛋白的制备方法,其特征在于,包括以下步骤:
表达载体的构建:全基因组合成含有CTB-Cap和乳酸菌表达载体pNZ8148的重组质粒pNZ8148-CTB-Cap,通过双酶切和PCR检测,筛选出含有CTB-Cap基因序列的克隆;
乳酸乳球菌表达系统的构建:通过电转化的方法,将获得的重组质粒pNZ8148-CTB-Cap转入乳酸菌感受态NZ9000;将转化后的菌体悬液涂布于含有氯霉素的GM17平板上,30℃培养;挑取单克隆,使用不同浓度的Nisin诱导表达CTB-Cap融合蛋白,超声破碎菌体;SDS-PAGE和Western blot检测确认阳性克隆。
4.CTB-Cap融合蛋白的编码基因,其碱基序列如SEQ ID NO:1所示。
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