猪瘟病毒E2(gp55)基因的克隆表达及其DNA疫苗的初步研究

用RTPCR方法从中国标准强毒株石门毒的细胞培养物中 扩增获得了其结构蛋白E2基因cDNA，将之克隆到pGEM5Z T载体，用双脱氧链终止法测定其核苷酸序列，并推导出其对应氨基酸序列，与几个代表毒株Alfort株、Brescia株和C株相应序列进行比较，所测核苷酸序列与各株的同源性分别为84.7%、92.6%和95.2%，氨基酸序列的同源性分别为89.4%，92.6%和94.6%；将此E2片段亚克隆至真核表达载体pcDNA3.1，构建表达CSFV E2蛋白的重组质粒pcE2，用脂质体转染法将pcE2导入cos7细胞进行瞬时表达，用针对E2蛋白的特异性单抗以间接免疫荧光法检测，结果E2蛋白在cos7细胞中获得了正确表达，随之将pcE2质粒DNA进行小鼠肌内接种免疫，ELISA法检测证实在免疫后2周和4 周的小鼠体内可诱导出较为明显的阳性血清，并高于E2单抗的阳性对照，病毒中和试验也表明DNA免疫后小鼠体内可诱导产生CSFV中和抗体；同时构建了能在昆虫细胞Sf9中表达GSTE2和GSTGFPE2融合蛋白的重组杆状病毒；上述研究结果为研制针对CSFV的DNA疫苗，亚单位疫苗及其诊断试剂打下了基础。

MOLECLONING AND EXPRKSSION OF E2 GENE OF THE CHINKSE CLASSICAL SWINE FEVER VIRUS(SHIMEN STRAIN) AND PRELIMINARY STUDIES OF ITS DNA VACCINE

A 1 1bp fragment of E2 gene of chinese classical swine fever virus(CSFV)Shimen strain, a standard virulent strain, was amplified by RT PCR from total RNA of cell cultures infected by CSFV, and cloned into pGEM T vector. the nucleotide sequence of this fragment was sequenced by Sanger's method and the amino acid sequence was deduced. Compared with the corresponding region of Alfort, Brescia and C strain of CSFV, the nucleotide sequence homology is 84 7%, 92 6% and 95 2% respectively, and the amino acid sequence 89 4%, 92 6% and 94 6%, respectively. we subcloned 1 1bp of E2 gene cDNA into baculovirus transfer vector and successfully constructed two recombinant baculoviruses expressing GST E2 and GST GFP E2 fusion protein respectively by homologous recombination in sf 9 cell. Furthermore, we also constructed recombinant eukaryotic expression vector pcE2 containing E2 gene in frame and transfected COS 7 cell by lipofectamine, the indirect immunofluorescence assay (IFA) showed that the expressed E2 protein can be recognized by E2 specific monoclnal antibody. the pcE2 DNA was directly injected into BALB/c mice intramusclarly(i.m) and the CSFV E2 specific antibodies was measured by enzyme linked immunosorbent assay(ELISA). the ELISA results indicated the E2 specific antibodies was induced in inoculated mice and virus neutralization assays also indicate single inoculations of plasmids expressing CSFV E2 glycoprotein raised neutralizing antibody in BALB/c mice. these results will be beneficial to investigate the possibility of DNA caccine against CSFV.