SARS-CoV S蛋白全长基因克隆及其表达的初步研究

目的:克隆表达SARS-CoVS蛋白,构建SARS全长基因疫苗。方法:从SARS病毒的总cDNA中以PCR方法扩增编码人SARS-CoVS1和S2蛋白的编码序列,再将二者分别克隆入真核表达载体pVAX1,构建重组表达载体。然后进一步将S1和S2连接到pVAX1中。酶切和测序鉴定阳性克隆。采用脂质体法转染VeroE6细胞,用Western检测S蛋白的表达。结果:PCR方法分别扩增出了1900bp和1800bp左右的基因片段,两片段插入pVAX1构建重组表达载体后,经序列测定证实该插入片段为SARS病毒S蛋白编码序列。将重组子转染VeroE6细胞,收集细胞总蛋白,Western检测获得特异蛋白带。结论:成功克隆并表达了SARS-CoVS蛋白,为其作为SARS基因疫苗研究打下基础。

Preliminary study on cloning and expression of full-length SARS-CoV S protein

AIM:To clone and express SARS CoV S protein and to construct SARS gene vaccine.MOTHODS:SARS CoV S1 and S2 gene were amplified from the cDNA of SARS CoV by PCR and the genes were respectively cloned into vector pVAX1 to construct recombinant expression vector. The two fragment of S1 and S2 were ligated into pVAX1 and the positive clones were identified by double digested and sequencing. The recombinant was transfected into Vero E6 cells by DOTAP reagent based on liposome technique, and then the expression of S protein was detected with Western blot.RESULTS:The gene fragments of about 1 900 bp and 1 800 bp were amplified by PCR.Following the two fragment cloned into the same vector pVAX1,sequencing results indicated that the sequence was as same as that of S derived from Tor2 strain except several site non frameshift mutation.After the recombinant was transfected into Vero E6 cells,Western blot showed specific protein band on membrane with anticipated molecular weight.