摘要
利用PCR技术,从含非洲猪瘟(ASFV)P72基因的克隆质粒BBBBPr4中扩增出1.94kb的P72基因。将P72基因和表达载体pET-30c(+)分别用限制性核酸内切酶FbaⅠ/XHoⅠ和BamHⅠ/XHoⅠ进行双酶切,然后在T4DNA连接酶作用下,将P72基因定向克隆至载体pET-30c(+)中相应位点上,并转化至宿主菌BL21(DE3)中,得到了重组菌株BL21(DE3)(pET-ASFVP72)。经PCR鉴定,构建的重组菌株中含有P72基因。重组菌株BL21(DE3)(pET-ASFVP72)经IPTG诱导后,其表达产物经SDS-PAGE和Westernblot分析,结果表明衣壳蛋白P72基因可以在受体菌中高效表达,达到了菌体总蛋白的34%,表达产物的分子量约为78Ku,并能被ASFV抗体所识别。
The 1.94 kb P72 gene was amplied from plasmid BBBBPr4 by polymerase chain reaction （PCR）. The PCR products were digested with restriction endonucleases Fba Ⅰ/XHo Ⅰ, and then P72 gene was inserted into the Barn H Ⅰ /Xho I site of pET-30c（＋） vector. Then the constructed recombinant plasmid was transformed into BL21 （DE3）, the recombinant strain BL21 （DE3）（pET-ASFVP72） was obtained. Its expressed product was about 34 % of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis. Its molecular weight was about 78 Ku. By Western blot we also know it can be recognized by the polyclonic antibody of ASFV.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2006年第3期267-270,共4页
Chinese Journal of Preventive Veterinary Medicine
