北京地区不同基因型Noro病毒的主要衣壳蛋白在大肠杆菌中的表达

Research of the E. coli expressed major capsid proteins from Noroviruses with different genotypes collected in Beijing area

目的对北京地区的不同基因型Noro病毒的主要衣壳蛋白（VP1）进行表达，得到Noro病毒抗原，为对Noro病毒做进一步的深入研究打下基础。方法用PCR分别从先前研究得到的含北京Noro病毒主要衣壳蛋白VP1编码基因的重组质粒pBST-CR2987（GⅡ-3）及pBST-CR2932（GⅡ4）中扩增得到VP1全基因，然后将目的基因亚克隆到表达载体pET-30a（＋）中，得到重组质粒，转化BL21（DE3）感受态细胞，利用异丙基-β-D-硫代半乳糖苷（IPTG）诱导蛋白表达。通过SDS-PAGE和Western blot检测蛋白的表达和抗原性，并对表达产物进行了纯化。结果（1）双酶切和测序证明得到了VP1的重组表达载体。CR2987 VP1基因全长1647 bp，含编码549个氨基酸的单一的开放读码框架（ORF）；CR2932 VP1基因全长1623 bp，含编码541个氨基酸的单一的ORF。（2）重组表达的VP1主要以包涵体的形式存在，在诱导后4—6h表达量最高。（3）重组表达的VP1能够被豚鼠抗Noro病毒VP1的特异性免疫血清及鼠抗His标签的抗体所识别。（4）纯化后的蛋白能够为人血清所识别。结论GⅡ-3型及GⅡ-4型Noro病毒北京地方株的VP1在原核体系中获得表达，表达的VP1可以被特异性免疫血清及人血清所识别，说明其具有抗原活性。

Objective To obtain the specific antigens of the expressed major capsid proteins from Norovirnses with different sub-genotypes in Beijing area. Methods The full-length genes of the major capsid proteins （VP1） were obtained through the amplification of the VP1 encoding gene in the recombinant plasmids pBST-CR2987 （ GⅡ -3） and pBST-CR2932 （ GⅡ -4）, which represented different Norovirns genotypes. The full-length genes were sub-cloned into the expression vector pET-30a（ ＋ ）, resulting in a recombinant plasmid, with which the BL21 competent cells were transformed, and the expression of the gene was induced by adding IPTG to the growth culture. The expression of the major capsid proteins were analyzed with Coomassie blue staining after SDS-PAGE, and assayed by Western blot with serum from human. Results （1） The major capsid protein genes of CR2987 and CR2932 were sub-cloned into expression vector pET-30a（ ＋ ）. The VP1 encoding genes were 1647 bp in length for CR2987 and 1623 bp for CR2932. The open reading frames （ORF） coded for 549 and 541 amino acids for these two proteins, respectively. （2）The expressed VPls were present primarily as inclusion bodies, and the maximal amount of the expressed proteins occurred at 4-6 h after IPTG induction. （3） These VPls could be recognized by specific immune serum against VP1 of Norovirns as well as His-tag antibody. Conclusion The VP1 s of CR2987 and CP,2932 are expressed in BL21 E. coli cells. The expressed VPls could react with specific immune serum against VP1 of Norovirns, indicating that the expressed VPls are of antigenicity.