Asia1型口蹄疫病毒分离株结构蛋白基因的克隆与表达

口蹄疫病毒结构蛋白氨基酸的变化是病毒抗原性变异的分子基础,大部分抗原表位位于主要的免疫原蛋白VP1上,部分非线性抗原表位位于VP2和VP3上。本研究首次成功测定了 Asia1 型口蹄疫病毒(YNBS/58)四种结构蛋白基因( p1 区)的核苷酸序列,全长 2199 个碱基,编码 733 个氨基酸,该基因与 Ind63/72、Pka3/54、Israel、China/99、C1/Germany、A22、ZIM7/83/2 毒株的 p1 基因核苷酸序列同源性分别为 88. 4%、86. 0%、89. 3%、68.6%、67.6%、66.8%、50.3%,推导的氨基酸序列同源性分别为 94.1%、93.2%、95.1%、79.9%、77.0%、76.5%、58.1%;将YNBS/58株与 Ind63/72、Pka3/54、Israel株的 vp1、vp2、vp3、vp4 基因和编码蛋白分别进行同源性比较,发现VP1的序列变异最大,VP2、VP3、VP4次之,且VP1的氨基酸变异主要集中在 42-50 位和 137-156 位。实现了YNBS/58株结构蛋白基因在大肠杆菌中的高效表达,其表达的融合蛋白以包涵体形式存在,分子量约为88kDa,占菌体总蛋白的16%左右,并利用镍柱对目的蛋白进行了纯化,纯度达 90%以上,本实验为进一步研究 A sia1型口蹄疫病毒的分子流行病学、p1基因及其编码蛋白的生物学功能奠定了基础。

Molecular Characteristics and Expression of Structural Protein Gene of Foot-and-Mouth Disease Virus Type Asia1

Variations in the amino acid sequence of Foot-and-mouth disease virus (FMDV) structural proteins are the molecular basis for the antigen diversity of the virus. Majority of antigenic sites for the virus neutralization are present on VP1, the major immunogenic protein. However, a few conformational epitopes are present on the structural proteins VP2 and VP3. The nucleotide sequence encoding all four structural proteins(P1 region) of FMDV type Asia1 YNBS/58 was determined. The P1 region of type Asia1 YNBS/58 is 2199 bp in length and codes for a polypeptide of 733 amino acids. The structural protein of vp4,vp2,vp3 and vp1 gene consists of 255,657,654,633 bp respectively. The nucleotide sequence identity of p1 gene between YNBS/58 and Ind63/72, Pka3/54, Israel, China/99, C1/Germany, A22, ZIM7/83/2 strains is 88.4%,86.0%,89.3%,68.6%,67.6%,66.8% and 50.3%, and the amino acid identity is 94.1%,93.2%,95.1%,79.9%,77.0%,76.5%,58.1%. The variations are unequally distributed among the four structural proteins of YNBS/58 and Ind 63/72, Pka3/54,Israel.The results showed that the variation in the derived amino acid sequence is the highest in VP1 followed by VP2,VP3 and VP4. The recombinant P1 protein was expressed in E.coli and was approximately 88 kDa in size, which accounted for 16% of total protein.The target protein was purified.