猪传染性胃肠炎病毒S基因全抗原位点片段及猪呼吸道冠状病毒缺失片段表达产物的反应原性比较

Comparsion of Antigenicity Between Expressed Proteins of The Fragment Including S Gene Whole Antigenic Sites and the Deleted Fragment in Porcine Respiratory Coronavirus of Transmissible Gastroeritis Virus

猪呼吸道冠状病毒(PRCV)是猪传染性胃肠炎病毒(TGEV)的自然缺失株,二者主要区别在于PRCV的S基因缺失B、C两个主要抗原位点。因此,利用RT-PCR技术分别扩增TGEV Purdue毒株S基因近N端的S1和S1-2两个片段,将之克隆到pGEX-KG原核表达载体的GST基因下游,经表达,获得大小约52kD和108kD两种融合蛋白,表达量分别占菌体总蛋白的45%和35%,主要以包涵体的形式存在。利用TGEV和PRCV两种阳性血清进行Western blot检测,当第一抗体为TGEV阳性血清时,可检测到52kD和108kD两条带;而当第一抗体为PRCV阳性血清时,只检测到108kD一条带。结果表明这两种融合蛋白具有良好的反应原性,且可用于TGEV和PRCV的鉴别诊断,为进一步研制开发TGEV和PRCV的鉴别诊断试剂盒奠定了基础。

The porcine respiratory coronavirus（PRCV） is a deletion mutant of transmissible gastroenteritis virus （TGEV） with altered respiratory tissue tropism. The important difference between them is that PRCV has a large deletion of two antigenic sites, B and C, in the 5＇region of the S gene. Two pairs of oligonucleotide primers appropriate for PCR amplification were designed based on the gene encoding the S protein of TGEV. The S1 （approximately 680bp, including B, C antigenic sites） and S1- 2 （approximately 2.2kb, including A, B, C, D antigenic sites） fragments were amplified by reverse transcription-polymerase chain reaction （RT-PCR） as- say. Two recombinant expressing vectors pGEX-S1 ,pGEX-S1 2 were constructed by insertion separately of the different S genes. Then the recombinant plasmids were transformed into E. coli BL21 competent cells. SDS- PAGE analysis revealed that we had obtained two kinds of fusion protein ,52kD and 108kD, after expression and the production of the proteins were 45 % and 35 % respectively. Using TGEV positive serum as first antibody in Western blot, two bands of 52kD and 108kD were detected; when using the PRCV positive serum as first antibody, only one band of 108kD was detected. The results showed that the fusion proteins have favorable reactinogenicity.