CN111662364A - 传染性法氏囊病病毒vp2蛋白及其编码基因与应用 - Google Patents
传染性法氏囊病病毒vp2蛋白及其编码基因与应用 Download PDFInfo
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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,具体是一种传染性法氏囊病病毒VP2蛋白及其编码基因与应用。传染性法氏囊病病毒VP2蛋白的氨基酸序列如SEQ ID NO:2所示。传染性法氏囊病病毒VP2蛋白的编码基因,其碱基序列如SEQ ID NO:1所示。本发明所述的传染性法氏囊病病毒VP2蛋白很好地利用了VP2和Pichia pastoris的优点,其编码基因可以在Pichia pastoris中高效表达,降低了生产成本,将此重组蛋白在制备传染性法氏囊病疫苗中的应用,具有很高的经济价值和巨大的社会效益。
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,具体是一种传染性法氏囊病病毒VP2蛋白及其编码基因与应用。
背景技术
传染性法氏囊病(Infectious Bursal Disease,IBD)在世界各地都有发生,给养鸡业造成了重大的经济损失。传染性法氏囊病病毒(Infectious Bursal Disease Virus,IBDV)是引发IBD的病原。目前,该病毒的中等毒力活疫苗、弱毒疫苗和灭活疫苗在IBDV的预防中被广泛地应用。然而,中等毒力活疫苗虽然能够抵抗IBDV超强毒株的攻击,但会对鸡的法氏囊产生一定的损伤。另外,使用活疫苗还存在病毒重组和毒力反强的风险。常规减毒活病毒疫苗的相关风险使得开发安全有效的新型疫苗成为必要。其中,亚单位疫苗已成为当前新型疫苗研究的热点。在IBDV的5种蛋白中,只有VP2和VP3是结构蛋白,共同构成病毒的核衣壳。VP2蛋白位于核衣壳表面是病毒最主要保护性抗原,也是IBDV亚单位疫苗开发的靶蛋白。
本发明通过毕赤酵母真核表达***对IBDV超强毒株UK661的VP2蛋白进行表达,用表达的VP2蛋白进行免疫保护试验,以期能为传染性法氏囊病亚单位疫苗的开发提供一定的理论依据。
发明内容
本发明利用传染性法氏囊病病毒VP2蛋白和Pichia pastoris的优点,提供了一种传染性法氏囊病病毒VP2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:传染性法氏囊病病毒VP2蛋白,其氨基酸序列如SEQ ID NO:2所示。
所述传染性法氏囊病病毒VP2蛋白在制备传染性法氏囊病疫苗中的应用。
本发明进一步提供了所述传染性法氏囊病病毒VP2蛋白的制备方法,包括以下步骤:
表达载体的构建:人工合成的VP2的基因序列上设计有限制性内切酶的酶切位点,将VP2的基因序列和表达质粒pwPICZalpha用同样的限制性内切酶酶切后连接在一起,通过双酶切检测和筛选含有VP2的基因序列的克隆;
酵母表达***的构建:使用限制性内切酶线性化重组表达质粒,通过电转化的方法,将VP2的基因序列整合到酵母的基因组中;将转化后的菌体悬液涂布于含有Zeocin的YPD平板上,30℃培养;挑取单单菌落,进行小剂量的诱导表达,SDS-PAGE检测表达上清确认阳性克隆;
重组蛋白的纯化和活性检测:挑选阳性克隆菌落进行大剂量的诱导表达,表达上清采用Protein Pure Ni-NTA 树脂纯化,通过SDS-PAGE和Western blot检测纯化的重组蛋白。
本发明进一步提供了传染性法氏囊病病毒VP2蛋白的编码基因,其碱基序列如SEQID NO:1所示。
所述传染性法氏囊病病毒VP2蛋白的编码基因是由编码传染性法氏囊病病毒VP2蛋白的cDNA序列、6个组氨酸cDNA序列和限制性内切酶Xho Ⅰ和EcoR Ⅰ位点的cDNA序列以及符合酵母表达偏好性的基因序列组成的。
本发明所述的传染性法氏囊病病毒VP2蛋白很好地利用了VP2和Pichia pastoris的优点,其编码基因可以在Pichia pastoris中高效表达,降低了生产成本,将此重组蛋白在制备传染性法氏囊病疫苗中的应用,具有很高的经济价值和巨大的社会效益。
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 是重组蛋白VP2的SDS-PAGE和Western blot分析图。左图为VP2蛋白考马斯亮蓝染色结果,右图为VP2蛋白用抗6×His抗体做Western blot结果。其中M为蛋白分子量标准(KDa),第1和4泳道为去糖基化酶处理后的VP2重组蛋白;2、3、5和6泳道为未经任何处理的VP2重组蛋白。
图2 是Ni-NTA Resin柱纯化重组蛋白VP2的SDS-PAGE分析图。其中M为蛋白分子量标准(KDa),P1-P8为平衡液;X1-X8为洗脱液。
图3 是重组蛋白VP2雏鸡后IBDV特异性抗体检测分析图。其中A:首免前,B:首免后一周,C:首免后两周,D:二免后一周,E:二免后两周。由图可以看出:表达的VP2蛋白配合佐剂免疫鸡,能够产生诱导机体产生IBDV抗体。
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(Luria-Bertani)液体培养基:取蛋白胨10g、酵母粉5g、NaCl 5g,溶解于800mLddH2O中,10M NaOH调节pH至7.0,定容至1000mL,高压灭菌(121℃,1.034×105Pa)20min,4℃保存。
(2)LB(Luria-Bertani)固体培养基:按每1000mL ddH2O加15g琼脂粉,向LB液体培养基中加入琼脂粉,高压灭菌,4℃保存。
(3)贮备液:
10×YNB(13.4%,含硫酸铵而不含氨基酸):将34g YNB和100g硫酸铵热溶于1000mLddH2O中,过滤除菌,4℃保存。
500×生物素(0.02%):20mg生物素溶于100mL ddH2O中,过滤除菌,4℃保存。
10×葡萄糖(20%):200g葡萄糖溶于1000mL ddH2O中,高压灭菌,4℃保存。
10×甲醇(5%甲醇):5mL甲醇与95mL ddH2O混匀,过滤除菌,4℃保存。
10×甘油(10%甘油):100mL甘油与900mL ddH2O混匀,过滤除菌或高压灭菌,室温保存。
1M磷酸氢二钾缓冲液,pH=7.0:132mL 1M K2HPO4与868mL 1M KH2PO4混合,高压灭菌,室温保存。
10×酸水解酪素(10%酸水解酪素):100g酸水解酪素溶于1000mL ddH2O中,高压灭菌,4℃保存。
(4)YPD培养基(1.0%酵母提取物、2.0%蛋白胨、2.0%葡萄糖):10g酵母提取物、20g蛋白胨溶于900mL ddH2O中,加入20g琼脂制YPD板,高压灭菌,再加100mL 10×葡萄糖,4℃保存备用。
(5)YPG培养基(1.0%酵母提取物、2.0%蛋白胨、1.0%甘油):10g酵母提取物、20g蛋白胨溶于900mL ddH2O中,高压灭菌,再加100mL 10×甘油,4℃保存备用。
(6)BMMYC培养基(1.34%YNB、4×10-5%生物素、0.5%甲醇、1.0%酵母提取物、2.0%蛋白胨、1.0%酸水解酪素):10g酵母提取物、20g蛋白胨溶于600mL ddH2O中,高压灭菌,冷却至室温,加100mL 1M磷酸氢二钾缓冲液,pH=7.0、100mL 10×YNB、2mL 500×生物素、100mL 10×甲醇、100mL 10×酸水解酪素混匀,4℃保存。
(7)缓冲液:Buffer(50 mM磷酸钠,0.3 M氯化钠,10 mM咪唑和10 mM pH 8.0Tris-HCl); Buffer(50 mM磷酸钠,0.3 M氯化钠,500mM咪唑和10 mM pH 8.0 Tris-HCl)。
2、VP2基因序列的优化和获得
根据NCBI登陆的VP2基因序列(AJ878898.1)和酵母菌密码子偏好性,对VP2的基因序列进行优化,为了便于纯化,在C端加入6个组氨酸(6×His tag),在序列的两端引入限制性内切酶Xho Ⅰ和EcoR Ⅰ位点。优化后的VP2基因序列由Invitrogen公司合成,其核苷酸序列如SEQ ID NO.2所示。
3、表达载体的构建
利用XhoⅠ和EcoRⅠ两个限制性内切酶对合成的VP2和质粒pwPICZalpha进行双酶切,酶切后产物进行琼脂糖电泳并采用胶回收试剂盒回收DNA片段并进行连接。
在无菌Eppendorf管中7μL双酶切的目的基因片段,1μL双酶切的质粒,1μL T4连接酶,1μL连接酶缓冲液,混匀,4℃连接12h;将10μL连接产物加入到90μL感受态大肠杆菌Trans 10中,混匀,42℃热激90s转化,加入400μL LB液体培养基,37℃振荡培养1h,将菌液涂布在Zeocin抗性的LB固体培养基上,37℃培养过夜。从转化的平板上挑取单菌落接种到含Zeocin的5mL LB液体培养基中,37℃过夜培养。用质粒提取试剂盒提取质粒。用XhoⅠ和EcoRⅠ对连接产物进行双酶切鉴定,验证基因片段正确***,该重组质粒命名为pwPICZalpha-VP2。
4、重组蛋白的表达
1)小剂量诱导表达
利用限制性内切酶SacⅠ将5-10μg重组质粒pwPICZalpha-VP2进行线性化,采用PCR产物纯化试剂盒回收线性化的pwPICZalpha-VP2,加入到90μL感受态毕赤酵母X-33中,混匀,冰浴5min后电击转化,加入1mL山梨醇后,30℃放置2h,将菌液涂布在Zeocin抗性的YPD固体培养基上,30℃培养3-4d。从转化的平板上挑取单菌落接种到含Zeocin的5mL YPD液体培养基中,30℃ 250rpm培养24h,3500rpm离心,弃上清后加入5mL YPG液体培养基,30℃ 250rpm培养24h,3500rpm离心弃掉上清,加入2mL BMMYC液体培养基,27℃ 225rpm培养48h,每隔12h补充一次甲醇,使甲醇浓度维持在0.5%。3500rpm离心后收集上清液,SDS-PAGE分析上清液。
2)大剂量诱导表达
挑取一个阳性菌落接种到YPD液体培养基中,30℃ 250rpm培养24h作为种子培养液,取13mL种子培养液接种到250mL YPD液体培养基中,30℃ 250rpm培养24h,30℃ 250rpm培养24h,3500rpm离心,弃上清后加入250mL YPG液体培养基,30℃ 250rpm培养24h,3500rpm离心弃掉上清,加入125mL BMMYC液体培养基,27℃ 225rpm培养48h,每隔12h补充一次甲醇,使甲醇浓度维持在0.5%。3500rpm离心后收集上清液,SDS-PAGE分析上清液。
5、重组蛋白Ni-NTA Resin柱纯化
在诱导表达的上清液中加入终浓度为50mM磷酸钠,0.3M氯化钠,10mM咪唑和10mM pH8.0 Tris-HCl,用0.45μm滤器过滤。取10mL Ni-NTA Resin柱装入XK16/20层析柱中,柱子装好后与恒流泵相连,用10倍体积的Buffer 平衡柱子,平衡好后上样,上样结束后用8倍体积的Buffer 冲洗柱子,再用8倍体积的Buffer Ⅱ洗脱目的蛋白并收集到8个不同的管中,整个过程的流速为5mL/min。用SDS-PAGE对纯化的蛋白进行分析,图2中的SDS-PAGE结果显示,目的蛋白集中在Buffer 的2号和3号管中。将含有目的蛋白的Buffer Ⅱ混合,装入3.5kDa透析袋中,在4℃条件下用含有20mM Tris-HCl pH 8.0,1mM EDTA pH8.0,5%甘油的透析液透析8h,每隔4h换一次透析液。
6、小鼠免疫保护试验
6.1 抗原制备
根据ISA 206佐剂使用说明书,30℃水浴加热纯化后的VP2蛋白和ISA 206佐剂,分别将不同浓度的纯化后的VP2蛋白加入等体积的ISA 206佐剂,室温下在涡旋振荡器上振荡10min,然后20℃条件下静置1 h,形成水包油包水型乳化疫苗。
6.2 小鼠试验分组及试验设计
将54只1日龄的SPF雏鸡,随机分成9个组,每组各6只。分别隔离饲养,使鸡自由采食饮水。具体分组与免疫情况如下:
(1)10 μg蛋白组:表达的VP2蛋白10 μg/只;
(2)40 μg蛋白组:表达的VP2蛋白40 μg/只;
(3)160 μg蛋白组:表达的VP2蛋白160 μg/只;
(4)10 μg蛋白+佐剂组:ISA 206佐剂和表达的VP2蛋白10 μg/只;
(5)40 μg蛋白+佐剂组:ISA 206佐剂和表达的VP2蛋白40 μg/只;
(6)160 μg蛋白+佐剂组:ISA 206佐剂和表达的VP2蛋白160 μg/只;
(7)B87疫苗组:IBDV中等毒力活疫苗(B 87株);
(8)PBS对照组:相同体积的PBS;
(9)空白对照组:不做任何处理。
将小鸡饲养至21日龄后进行首次免疫,1-8组均采用腿部肌肉注射的方式,每只鸡注射体积为300 μL。
6.3 IBDV抗体检测
分别在首次免疫前(0 d)、首次免疫一周后(7 d)、首次免疫二周后(14 d)、二次免疫一周后(21 d)和二次免疫二周后(28 d)对1-8组鸡进行翅下静脉采血,分离血清备用。
在650 nm波长处测吸光值,根据下列方法计算抗体滴度:S/P值=(样品平均值-阴性对照平均值)/(阳性对照平均值-阴性对照平均值)
抗体滴度与S/P的关系:Log10滴度=1.09(Log10 S/P)+3.36
样品的抗体滴度大于396时判断为IBD阳性,说明接种过IBD疫苗或感染了IBDV。
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>传染性法氏囊病病毒VP2蛋白及其编码基因与应用
<160>2
<210>1
<211>1386
<212>DNA
<213>人工序列
<220>
<221>CDS
<223>传染性法氏囊病病毒VP2蛋白的编码基因
<222>(1)…(1386)
<400>1
CTCGAGAAGA GAGAGGCTGA AGCTCAACAA ATCGTTCCAT TCATCAGATC 50
TTTGTTGATG CCAACTACTG GTCCAGCTTC TATCCCAGAC GACACTTTGG 100
AAAAGCACAC TTTGAGATCT GAAACTTCTA CTTACAACTT GACTGTTGGT 150
GACACTGGTT CTGGTTTGAT CGTTTTCTTC CCAGGTTTCC CAGGTTCTAT 200
CGTTGGTGCT CACTACACTT TGCAATCTAA CGGTAACTAC AAGTTCGACC 250
AAATGTTGTT GACTGCTCAA AACTTGCCAG CTTCTTACAA CTACTGTAGA 300
TTGGTTTCTA GATCTTTGAC TGTTAGATCT TCTACTTTGC CAGGTGGTGT 350
TTACGCTTTG AACGGTACTA TCAACGCTGT TACTTTCCAA GGTTCTTTGT 400
CTGAATTGAC TGACGTTTCT TACAACGGTT TGATGTCTGC TACTGCTAAC 450
ATCAACGACA AGATCGGTAA CGTTTTGGTT GGTGAAGGTG TTACTGTTTT 500
GTCTTTGCCA ACTTCTTACG ACTTGGGTTA CGTTAGATTG GGTGACCCAA 550
TCCCAGCTAT CGGTTTGGAC CCAAAGATGG TTGCTACTTG TGACTCTTCT 600
GACAGACCAA GAGTTTACAC TATCACTGCT GCTGACGACT ACCAATTCTC 650
TTCTCAATAC CAAGCTGGTG GTGTTACTAT CACTTTGTTC TCTGCTAACA 700
TCGACGCTAT CACTTCTTTG TCTATCGGTG GTGAATTGGT TTTCCAAACT 750
TCTGTTCAAG GTTTGATCTT GGGTGCTACT ATCTACTTGA TCGGTTTCGA 800
CGGTACTGCT GTTATCACTA GAGCTGTTGC TGCTGACAAC GGTTTGACTG 850
CTGGTACTGA CAACTTGATG CCATTCAACA TCGTTATCCC AACTTCTGAA 900
ATCACTCAAC CAATCACTTC TATCAAGTTG GAAATCGTTA CTTCTAAGTC 950
TGGTGGTCAA GCTGGTGACC AAATGTCTTG GTCTGCTTCT GGTTCTTTGG 1000
CTGTTACTAT CCACGGTGGT AACTACCCAG GTGCTTTGAG ACCAGTTACT 1050
TTGGTTGCTT ACGAAAGAGT TGCTACTGGT TCTGTTGTTA CTGTTGCTGG 1100
TGTTTCTAAC TTCGAATTGA TCCCAAACCC AGAATTGGCT AAGAACTTGG 1150
TTACTGAATA CGGTAGATTC GACCCAGGTG CTATGAACTA CACTAAGTTG 1200
ATCTTGTCTG AAAGAGACAG ATTGGGTATC AAGACTGTTT GGCCAACTAG 1250
AGAATACACT GACTTCAGAG AATACTTCAT GGAAGTTGCT GACTTGAACT 1300
CTCCATTGAA GATCGCTGGT GCTTTCGGTT TCAAGGACAT CATCAGAGCT 1350
TTGAGAAGAC ACCACCACCA CCACCACTAA GAATTC 1386
<210>2
<211>457
<212>PRT
<213>人工序列
<223>传染性法氏囊病病毒VP2蛋白
<400>2
KREAEAQQIV PFIRSLLMPT TGPASIPDDT LEKHTLRSET STYNLTVGDT 50
GSGLIVFFPG FPGSIVGAHY TLQSNGNYKF DQMLLTAQNL PASYNYCRLV 100
SRSLTVRSST LPGGVYALNG TINAVTFQGS LSELTDVSYN GLMSATANIN 150
DKIGNVLVGE GVTVLSLPTS YDLGYVRLGD PIPAIGLDPK MVATCDSSDR 200
PRVYTITAAD DYQFSSQYQA GGVTITLFSA NIDAITSLSI GGELVFQTSV 250
QGLILGATIY LIGFDGTAVI TRAVAADNGL TAGTDNLMPF NIVIPTSEIT 300
QPITSIKLEI VTSKSGGQAG DQMSWSASGS LAVTIHGGNY PGALRPVTLV 350
AYERVATGSV VTVAGVSNFE LIPNPELAKN LVTEYGRFDP GAMNYTKLIL 400
SERDRLGIKT VWPTREYTDF REYFMEVADL NSPLKIAGAF GFKDIIRALR 450
RHHHHHH 457
Claims (4)
1.传染性法氏囊病病毒VP2蛋白,其氨基酸序列如SEQ ID NO:2所示。
2.权利要求1所述传染性法氏囊病病毒VP2蛋白在制备传染性法氏囊病疫苗中的应用。
3.权利要求1所述传染性法氏囊病病毒VP2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:人工合成的VP2的基因序列上设计有限制性内切酶的酶切位点,将VP2的基因序列和表达质粒pwPICZalpha用同样的限制性内切酶酶切后连接在一起,通过双酶切检测和筛选含有VP2的基因序列的克隆;
酵母表达***的构建:使用限制性内切酶线性化重组表达质粒,通过电转化的方法,将VP2的基因序列整合到酵母的基因组中;将转化后的菌体悬液涂布于含有Zeocin的YPD平板上,30℃培养;挑取单单菌落,进行小剂量的诱导表达,SDS-PAGE检测表达上清确认阳性克隆;
重组蛋白的纯化和活性检测:挑选阳性克隆菌落进行大剂量的诱导表达,表达上清采用Protein Pure Ni-NTA 树脂纯化,通过SDS-PAGE和Western blot检测纯化的重组蛋白。
4.传染性法氏囊病病毒VP2蛋白的编码基因,其碱基序列如SEQ ID NO:1所示。
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