构建含微小隐孢子虫病毒L-dsRNA基因的原核表达载体并表达,表达产物为进一步研究该蛋白的功能及特性提供材料。从微小隐孢子虫病毒L-dsRNA基因重组pMD-18T质粒中经H indⅢ和BamHⅠ双酶切回收目的片段,定向亚克隆入高效原核表达载体pET-2...构建含微小隐孢子虫病毒L-dsRNA基因的原核表达载体并表达,表达产物为进一步研究该蛋白的功能及特性提供材料。从微小隐孢子虫病毒L-dsRNA基因重组pMD-18T质粒中经H indⅢ和BamHⅠ双酶切回收目的片段,定向亚克隆入高效原核表达载体pET-28b(+)中,构建重组表达质粒,并转化入DE3菌中。经IPTG诱导表达,SDS-PAGE分析表达蛋白。经PCR、酶切鉴定表明,构建了开放阅读框完整的微小隐孢子虫病毒L-dsRNA基因的原核表达质粒pET-L-dsRNA,经IPTG诱导后菌体裂解液上清SDS-PAGE分析显示与预期大小相符的约62000 M r特异蛋白条带。成功的在原核表达系统中表达了微小隐孢子虫病毒L-dsRNA基因蛋白,可用于进一步研究该蛋白的功能和特性。展开更多
Avian infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).In the study,according to IBV gene sequences published in Gen Bank,specific primers were designed to ...Avian infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).In the study,according to IBV gene sequences published in Gen Bank,specific primers were designed to clone N gene by RT-PCR,and this gene was inserted into p ET-30a(+)vector resulting in a prokaryotic expression plasma p ET-30a-N.The results of SDS-PAGE and Western Blot analysis showed that the recombinant protein was expressed successfully and had good reactivity with IBV positive serum.Using purified recombinant N protein as a coating antigen,the indirect ELISA protocol was established and optimized,in which N protein was 2.5μg·m L^-1 of concentration,sample serum of 1:40 dilution.For clinical specimen,the IBV antibodies could be detected by this method efficiently and got nearly the same results as those of IBV-mediated ELISA.It would provide a good tool for rapid diagnosis and epidemiological study of avian infectious bronchitis.展开更多
文摘构建含微小隐孢子虫病毒L-dsRNA基因的原核表达载体并表达,表达产物为进一步研究该蛋白的功能及特性提供材料。从微小隐孢子虫病毒L-dsRNA基因重组pMD-18T质粒中经H indⅢ和BamHⅠ双酶切回收目的片段,定向亚克隆入高效原核表达载体pET-28b(+)中,构建重组表达质粒,并转化入DE3菌中。经IPTG诱导表达,SDS-PAGE分析表达蛋白。经PCR、酶切鉴定表明,构建了开放阅读框完整的微小隐孢子虫病毒L-dsRNA基因的原核表达质粒pET-L-dsRNA,经IPTG诱导后菌体裂解液上清SDS-PAGE分析显示与预期大小相符的约62000 M r特异蛋白条带。成功的在原核表达系统中表达了微小隐孢子虫病毒L-dsRNA基因蛋白,可用于进一步研究该蛋白的功能和特性。
基金Supported by the National Natural Science Foundation of China(31172295,31272569)。
文摘Avian infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).In the study,according to IBV gene sequences published in Gen Bank,specific primers were designed to clone N gene by RT-PCR,and this gene was inserted into p ET-30a(+)vector resulting in a prokaryotic expression plasma p ET-30a-N.The results of SDS-PAGE and Western Blot analysis showed that the recombinant protein was expressed successfully and had good reactivity with IBV positive serum.Using purified recombinant N protein as a coating antigen,the indirect ELISA protocol was established and optimized,in which N protein was 2.5μg·m L^-1 of concentration,sample serum of 1:40 dilution.For clinical specimen,the IBV antibodies could be detected by this method efficiently and got nearly the same results as those of IBV-mediated ELISA.It would provide a good tool for rapid diagnosis and epidemiological study of avian infectious bronchitis.
