摘要
本试验利用PCR技术,扩增得到猪瘟病毒(CSFV)C株E2基因的主要抗原片段A/D区549bp,将其酶切纯化后,与已酶切纯化的pET-32a进行16℃过夜连接,转化大肠杆菌BL21。从转化成功的氨苄培养板挑取合适菌落进行PCR,挑取目的条带正确的菌落加至LB培养基进行过夜培养后送测序。将测序正确的菌液划板,挑菌培养并用IPTG进行诱导表达。得到的蛋白进行SDS-PAGE,在约39ku位置有蛋白条带。将蛋白纯化后,分别利用自制兔抗CSFV阳性血清及临床检测阳性的猪抗CSFV阳性血清进行Western blotting检测,可见在39ku处有单一条带,表明表达的蛋白具有免疫原性。本试验结果为下一步ELSIA试剂盒的组装奠定基础,方便对猪场免疫猪群猪瘟抗体水平进行监控。
A truncated gene encoding the A/D antigenic domains （549 bp） of E2 in classical swine fever virus （CSFV） was amplified by RT PCR from the genomic RNA of CSFV C strain, cloned into pET 32a expression vector to obtain recombinant pET-32a-CSFV-E2, and then sequenced. The result was the right interested gene. The recombinant plasmid was transformed in- to BL21 and induced with isopropyl-β-D-thiogalactopyranoside （IPTG）. After expression,we could see our target band about 39 ku with SDS-PAGE. To purify the His-trap protein in different conditions,and we could get the best one with the most produc- tive target protein. Western blotting indicated that the expressed antigen protein could be recognized by the positive sera of CS FV （the sera from our homemade anti rabbit one and the clinical swine one）. It would benefit the creation of ELISA kit and re- search on the antibody levels of pigs.
出处
《中国畜牧兽医》
CAS
北大核心
2014年第5期12-16,共5页
China Animal Husbandry & Veterinary Medicine
基金
公益性行业(农业)科研专项(201203056)
国家基金面上项目(31172321)
广东省高等学校科技创新(重点)项目(2012CXZD0013)
关键词
猪瘟病毒C株
A
D主要抗原区
截短表达
classical swine fever virus （CSFV） C strain
A/D antigen domains
truncated expression
