【摘要】 通过RT-PCR扩增了编码猪瘟病毒兔化弱毒囊膜糖蛋白E2基因;另行设计引物,扩增其中一段所编码的蛋白质抗原性好且适于在大肠杆菌(Escherichiacoli)中表达的基因(E2蛋白A/D抗原区),构建了不同的重组原核表达载体(pTH-E2来源于pThioHisB载体;pET-E2来源于pET-32a(+)),并对其表达特性进行了研究。将其分别转化到相应的宿主菌中,构建成2株重组工程菌(TOPE2含有pTH-E2;BL21E2含有pET-E2)。结果表明,两种重组菌都获得了高效表达,但BL21E2表达量明显高于TOPE2。另一重要发现是,在E2蛋白上,除了690~812位肽段,所选肽段在大肠杆菌中表达后,也能和抗猪瘟病毒高免血清反应。更多还原

【Abstract】 Two prokaryotic expression systems, pThioHisB and pET-32a(+) were int roduced to compare their characteristics in expressing the E2 protein of HCLV(ho g cholera lapinised virus). The virions of HCLV in cell culture supernatant was concentrated using PEG6000 and then centrifugation. Two pairs of primers were de signed, one to amplify the full gene encoding E2 from the virus genome by RT-PCR , and another to amplify the specific gene encoding the A/D antigenic domains in E2 protein from the RT-PCR product. This PCR product was digested with SalⅠand PstⅠ and inserted into pThioHisB to get pTH-E2 recombinant plasmid. By digesti on with BglⅡ, the gene for A/D antigenic domains was inserted into pET-32a(+) t o get pET-E2. The two recombinant vectors were transformed into their competent cells, respectively to get TOPE2 (containing pTH-E2) and BL21E2 (containing pET-E2) recombinant bacteria. These two strains of bacteria were induced with IPTG a nd an SDS-PAGE analysis showed that they could both produce recombinant E2 prote in in a high level. Thin layer chromatography showed that the recombinant protei n accounted for 17.6% (TOPE2) and 41.8% (BL21E2) of gross bacteria protein. The expressed product of BL21E2 was analyzed by Western-blot, useing rabbit-anti-HCL V high-immune serum as a reagent. A specific band could be found at the expected position on the NC film. As a result, the pET system seems more suitable for E2 expression. Western-blot showed that the recombinant protein had good antigenic ity. Therefore, it might serve as a competent antigen for the detection of swine s’ antibody against HCLV in later research. Another important finding was that, besides 690~812 aminoacid on the E2 protein, there existed another region, maybe more, containing linear epitopes that could react with anti-HCLV antibody effec tively.更多还原