山羊痘病毒南疆株P32基因的原核表达及其结构分析
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摘要
为了研究新疆南疆分离的山羊痘病毒P32基因的特征,试验通过PCR扩增得到P32基因及其膜外区P32_(1～282)基因和膜外亲水区P32_(20～270)基因,分别构建了表达载体(pET42b-P32、pET42b-P32_(1～282)和pET28a-P32_(20～270))并进行了原核表达,对其核苷酸序列的同源性、遗传进化关系及推测的氨基酸序列、蛋白质二级结构进行了预测分析。结果表明:山羊痘病毒南疆株P32基因发生了较大突变,相对对照在第100～105位增加了6个核苷酸,编码K、N两个氨基酸,全长达到975 bp,编码324个氨基酸,与Gansu 2009株相似。跨膜分析显示P32蛋白膜外区为1～282位氨基酸,跨膜区为283～305位氨基酸,膜内区为306～324位氨基酸。二级结构分析发现其分子两端呈明显的疏水结构,抗原性较弱,而中间区域有较强的亲水性和较强的抗原性。蛋白质表达发现其全长在大肠杆菌中几乎没有表达,其膜外区可以表达,但表达量较低,难以纯化。说明山羊痘病毒P32蛋白是一个典型的膜蛋白,全长较难表达纯化,南疆株P32蛋白也具有这样的特性。
To study the characteristic of P32 gene of Goatpox virus separated from the Southern Xinjiang,PCR amplifying method was used to obtain P32 gene,its extracellular region P32_(1-282) and its extracellular hydrophilic region P32_(20-270). Expression vectors pET42 b-P32,pET42 b-P32_(1-282 )and pET28 a-P32_(20-270) were constructed respectively and were expressed prokaryoticly. The predicted nucleotide sequence's homology and genetic relationship,and its deduced amino acid sequence and secondary protein structure were analyzed. The results showed that there was a big mutation in P32 gene of Goatpox virus Nanjiang strain. Compared with control sequence,added 6 nucleotides were inserted at position 100-105 which encoded two amino acids K and N. The full gene length was 975 bp and it encoded 324 amino acids(AA),which was similar with Gansu2009 strain. Transmembrane analysis showed that extracellular region of P32 protein located at position 1-282 AA,and transmembrane region at position 283-305 AA,intramembrane region at position 306-324 AA. Secondary structure analysis showed that both ends of the molecule presented a clear hydrophobic structure and weak antigenicity,while the intermediate region was strongly hydrophilic and had strong antigenicity. Protein expression showed that the full-length protein had almost no expression in Escherichia coli,and extracellular region was expressed in the form of inclusion body but with low quantity and difficult purification. The results suggested that P32 protein of Goatpox virus was a typical membrane protein;the full-length protein had difficult expression and purification;and P32 protein of Nanjiang strain also had such features.
To study the characteristic of P32 gene of Goatpox virus separated from the Southern Xinjiang,PCR amplifying method was used to obtain P32 gene,its extracellular region P32_(1-282) and its extracellular hydrophilic region P32_(20-270). Expression vectors pET42 b-P32,pET42 b-P32_(1-282 )and pET28 a-P32_(20-270) were constructed respectively and were expressed prokaryoticly. The predicted nucleotide sequence's homology and genetic relationship,and its deduced amino acid sequence and secondary protein structure were analyzed. The results showed that there was a big mutation in P32 gene of Goatpox virus Nanjiang strain. Compared with control sequence,added 6 nucleotides were inserted at position 100-105 which encoded two amino acids K and N. The full gene length was 975 bp and it encoded 324 amino acids(AA),which was similar with Gansu2009 strain. Transmembrane analysis showed that extracellular region of P32 protein located at position 1-282 AA,and transmembrane region at position 283-305 AA,intramembrane region at position 306-324 AA. Secondary structure analysis showed that both ends of the molecule presented a clear hydrophobic structure and weak antigenicity,while the intermediate region was strongly hydrophilic and had strong antigenicity. Protein expression showed that the full-length protein had almost no expression in Escherichia coli,and extracellular region was expressed in the form of inclusion body but with low quantity and difficult purification. The results suggested that P32 protein of Goatpox virus was a typical membrane protein;the full-length protein had difficult expression and purification;and P32 protein of Nanjiang strain also had such features.
引文
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[12] 萨姆不鲁克 J,弗里奇 E F,曼尼阿蒂斯 T.分子克隆实验指南[M].2版.金冬雁,等译.北京:科学出版社,1992.
[13] 陈宏伟,刘涛,宋书婷,等.山羊痘病毒毒力因子KLP1的表达及多克隆抗体的制备[J].黑龙江畜牧兽医,2018(03):15-19.
[14] 朱立平,陈学清.免疫学常用实验方法[M].北京:人民军医出版社, 2000.
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[16] 程振涛.山羊痘病毒某些生物学特性及其主要结构蛋白P32基因的研究[D].贵阳:贵州大学, 2009.
[17] 丰素兰.羊痘病毒P32基因的克隆、表达及PCR诊断方法建立[D].北京:中国农业大学,2005.
[2] CRAN V M. Control of capripoxvirus infection [J].Vaccine,1993,11(3):1275-1279.
[3] 赵志荀,王建科,张强.羊痘病毒P32蛋白的研究进展[J].中国畜牧兽医,2009,36(10):129-132.
[4] 朱学亮,张强,杨帆,等.羊痘病毒载体研究进展[J].中国畜牧兽医,2009,36(5):98-101.
[5] CHAND P, KITCHING R P, BLACK D N, et al. Western blot analysis of virus-specific antibody responses for capripox and contagious pustular dermatitis viral infections in sheep[J]. Epidemiol Infect, 1994, 113(2): 377-385.
[6] 张前群,马承俊.山羊痘暴发流行的防制与思考[J].畜禽业,2004(9):20.
[7] 郭巍.羊痘病毒P32基因的克隆与表达[D].哈尔滨:东北农业大学,2004.
[8] 郭巍,相文华,李一经.羊痘病毒P32蛋白的表达[J]. 中国兽医杂志,2005,41(3):8-10.
[9] CRAN V M, TIMMS C P, CHAND P, et al. Protection of goats against capripox using a subunit vaccine[J]. Vet Rec,1994,135(18): 434-436.
[10] 郭巍,陈杰,李一经,等.羊痘病毒P32蛋白基因的克隆及其杆状病毒表达载体的构建[J].中国预防兽医学报,2004,26(4): 270-272.
[11] HEINE H G, STEVENTS M P, FOORD A J, et al. A capripoxvirus detection PCR and antibody ELISA based on the major antigen P32,the homolog of the vaccinia virus H3L gene [J]. J Immunol Methods, 1999,227(1/2): 187-196.
[12] 萨姆不鲁克 J,弗里奇 E F,曼尼阿蒂斯 T.分子克隆实验指南[M].2版.金冬雁,等译.北京:科学出版社,1992.
[13] 陈宏伟,刘涛,宋书婷,等.山羊痘病毒毒力因子KLP1的表达及多克隆抗体的制备[J].黑龙江畜牧兽医,2018(03):15-19.
[14] 朱立平,陈学清.免疫学常用实验方法[M].北京:人民军医出版社, 2000.
[15] 马维民.羊痘病毒结构蛋白P32基因的克隆及其在大肠埃希氏菌中的表达[D].兰州:甘肃农业大学,2006.
[16] 程振涛.山羊痘病毒某些生物学特性及其主要结构蛋白P32基因的研究[D].贵阳:贵州大学, 2009.
[17] 丰素兰.羊痘病毒P32基因的克隆、表达及PCR诊断方法建立[D].北京:中国农业大学,2005.