PEDV分离株S1基因的重组杆状病毒真核表达

doi:10.16431/j.cnki.1671-7236.2019.03.023

摘要/Abstract

摘要：

为了研究猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）分离株（PEDV/LA/2014/02）纤突蛋白（S1）的真核表达及其反应原性，本研究利用杆状病毒真核表达系统表达出重组His-PEDV-S1蛋白。利用在线软件分析PEDV S1基因在sf9细胞内的稀有密码子，经优化密码子后的基因进行人工合成，合成后的PEDV S1基因被克隆至杆状病毒的穿梭载体（pFastBac HT A）中，转化大肠杆菌DH10Bac感受态细胞进行重组，PCR方法验证后将重组成功的杆状病毒基因组转染sf9细胞，获得包装成功的杆状病毒，该病毒进一步接种sf9细胞，显微镜观察重组病毒引起的细胞病变，RT-PCR方法验证PEDV S1基因的mRNA表达水平，SDS-PAGE、Western blotting方法验证重组PEDV S1蛋白的表达及其反应原性。结果显示，试验成功构建了重组穿梭质粒pFastBac HT A-PEDV-S1（pSL598），成功包装表达了PEDV S1的重组杆状病毒，重组杆状病毒能使sf9细胞出现细胞变大、胞内有空泡等典型病变，PEDV S1基因的mRNA获得表达，重组蛋白His-PEDV-S1在sf9细胞中得到表达，蛋白质大小为83 ku左右，主要存在于细胞沉淀中，表达的重组蛋白能与小鼠抗His抗体和猪抗PEDV阳性血清反应，说明该蛋白具有较好的反应原性。本研究为研制PEDV新流行毒株新型亚单位疫苗和防控该毒株的流行提供了材料。

关键词: 猪流行性腹泻病毒(PEDV); 纤突蛋白(S1); 杆状病毒表达系统; 真核表达

Abstract:

In order to study the eukaryotic expression and reactivity of porcine epidemic diarrhea virus (PEDV) isolated strain (PEDV/LA/2014/02) S1 gene,the recombinant protein (His-PEDV-S1) was expressed by the baculovirus expression system.The rare codon of PEDV S1 gene in sf9 cells was analyzed using online software,and the optimized gene was synthetized by biotechnology company.PEDV S1 gene were cloned into the baculovirus shuttle vector (pFastBac HT A),the PEDV S1 gene was recombined into baculovirus genome in Esherichia coli DH10Bac cells and identied by PCR.The PEDV-S1 recombinant successfully baculovirus genome was transfected into sf9 cells.The packaging viruses were inoculated into sf9 cells,the cytopathic effect was observed by optical microscope.The mRNA level of PEDV S1 gene in sf9 cells was analyzed by RT-PCR.The expression and antigenicity of recombinant PEDV S1 were verificated by SDS-PAGE and Western blotting.The results showed that the pFastBac HT A-PEDV-S1 (pSL598) was successfully constructed,the recombinant baculovirus expressing His-PEDV-S1 was packaged,the sf9 cells became big and round,the vacuoles appeared in cytoplasm after baculovirus infection.The mRNA of PEDV S1 gene was expressed in baculovirus infected cells.The recombinant protein His-PEDV-S1 was mainly expressed in sf9 cells precipitation,and the size of the protein was about 83 ku.The His-PEDV-S1 had good reactivity of mouse anti-His antibody and swine anti-PEDV positive antisera.This study provided materials for the development of a new subunit vaccine for the new PEDV strain and the prevention and control of PEDV strain.

Key words: porcine epidemic diarrhea virus (PEDV); spike protein (S1); baculovirus expression system; eukaryotic expression

中图分类号:

S852.65⁺9.6

何海健, 吴瑗, 王鲁彦, 李群景, 巴少波, 石林, 邵春艳, 孙静, 姜胜, 王晓杜. PEDV分离株S1基因的重组杆状病毒真核表达[J]. 《中国畜牧兽医》, 2019, 46(3): 832-839.

HE Haijian, WU Yuan, WANG Luyan, LI Qunjin, BA Shaobo, SHI Lin, SHAO Chunyan, SUN Jing, JIANG Sheng, WANG Xiaodu. Eukaryotic Expression of Recombinant Baculovirus of PEDV Isolated Strain S1 Gene[J]. , 2019, 46(3): 832-839.

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