中国预防兽医学报
中國預防獸醫學報
중국예방수의학보
CHINESE JOURNAL OF PREVENTIVE VETERINARY MEDICINE
2010年
2期
126-130
,共5页
李晶%余兴龙%李润成%黄泽彬%葛猛%向卫军%李薇%王亚
李晶%餘興龍%李潤成%黃澤彬%葛猛%嚮衛軍%李薇%王亞
리정%여흥룡%리윤성%황택빈%갈맹%향위군%리미%왕아
猪巨细胞病毒%gB基因%原核表达%间接ELISA
豬巨細胞病毒%gB基因%原覈錶達%間接ELISA
저거세포병독%gB기인%원핵표체%간접ELISA
porcine cytomegalovirus%glycoprotein B gene%prokaryotic expression%indirect ELI-SA
为表达猪巨细胞病毒(PCMV)gB基因,本研究根据GenBank登录的PCMV gB基因序列设计1对引物,以感染PCMV猪肺细胞总DNA为模板,采用PCR扩增得到gB基因片段,克隆于pMD-18T并进行核苷酸序列测定.利用DNAStar分析gB蛋白的抗原表位,选择抗原表位富集的2个基因片段(命名为PCMVgB1和PCMVgB2)分别克隆到原核表达载体pET-32a(+)中,构建重组表达质粒并转化Rosetta(DE3)宿主菌.结果显示:扩增的gB基因与GenBank登录的PCMV gB基因的核苷酸同源性在97.6%～98.9%之间,推导的氨基酸同源性在97%～98.6%之间.经IPTG诱导的含pET-gB1和pET-gB2重组质粒的宿主菌可表达重组gB1和gB2蛋白,SDS-PAGE显示分子量约为62ku和36 ku.Western blot和间接ELISA结果表明,重组gB1和gB2蛋白均具有反应原性.
為錶達豬巨細胞病毒(PCMV)gB基因,本研究根據GenBank登錄的PCMV gB基因序列設計1對引物,以感染PCMV豬肺細胞總DNA為模闆,採用PCR擴增得到gB基因片段,剋隆于pMD-18T併進行覈苷痠序列測定.利用DNAStar分析gB蛋白的抗原錶位,選擇抗原錶位富集的2箇基因片段(命名為PCMVgB1和PCMVgB2)分彆剋隆到原覈錶達載體pET-32a(+)中,構建重組錶達質粒併轉化Rosetta(DE3)宿主菌.結果顯示:擴增的gB基因與GenBank登錄的PCMV gB基因的覈苷痠同源性在97.6%～98.9%之間,推導的氨基痠同源性在97%～98.6%之間.經IPTG誘導的含pET-gB1和pET-gB2重組質粒的宿主菌可錶達重組gB1和gB2蛋白,SDS-PAGE顯示分子量約為62ku和36 ku.Western blot和間接ELISA結果錶明,重組gB1和gB2蛋白均具有反應原性.
위표체저거세포병독(PCMV)gB기인,본연구근거GenBank등록적PCMV gB기인서렬설계1대인물,이감염PCMV저폐세포총DNA위모판,채용PCR확증득도gB기인편단,극륭우pMD-18T병진행핵감산서렬측정.이용DNAStar분석gB단백적항원표위,선택항원표위부집적2개기인편단(명명위PCMVgB1화PCMVgB2)분별극륭도원핵표체재체pET-32a(+)중,구건중조표체질립병전화Rosetta(DE3)숙주균.결과현시:확증적gB기인여GenBank등록적PCMV gB기인적핵감산동원성재97.6%～98.9%지간,추도적안기산동원성재97%～98.6%지간.경IPTG유도적함pET-gB1화pET-gB2중조질립적숙주균가표체중조gB1화gB2단백,SDS-PAGE현시분자량약위62ku화36 ku.Western blot화간접ELISA결과표명,중조gB1화gB2단백균구유반응원성.
The glycoprotein B (gB) gene encoding structural protein of porcine cytomegalovirus (PCMV) was amplified by PCR using primers derived from the published PCMV gB gene sequence, cloned and sequenced. The gB gene shared 97.6%-98.9% sequence identity at nucleotide level and 97%-98.6% identity at amino acid level with PCMV gB reference genes in GenBank. Two immunodominant regions of PCMV gB1 and PCMV gB2 were identified by DNAStar and were subcloned into prokaryotic expression vector pET-32a(+). The resulting plasmids of pET-gB1 and pET-gB2 were transformed into E.coli competent Rosetta, respectively, and induced by IPTG. Recombinant protein of 62 kua (gB1) protein and a 36 kua (gB2) protein were expressed and detected by SDS-PAGE. Western blot and indirect ELISA analysis showed that both recombinant proteins could specifically react with antiserum against PCMV.
