摘要
目的对猪瘟病毒石门株E2基因进行原核表达,以期获得可溶性表达产物,为检测猪瘟抗体的ELISA试剂盒的研制奠定基础。方法用PCR技术扩增了重组S21质粒载体上的猪瘟病毒石门株E2基因的4个主要抗原结构域ABCD,A1A2,B和C。分别将4个片段克隆于pMAL-p2X载体中,经PCR、双酶切和测序鉴定,E2基因的4个主要抗原结构域片段的位置、大小和读码框均正确。将4个片段分别转化到表达菌TB1、BL21、BL21-CodonPlus(DE3)-RP和BL21(DE3)中,共得到16株重组表达菌,用IPTG进行诱导表达,对表达产物进行SDS-PAGE电泳和免疫印迹分析。结果E2基因的4个主要抗原结构域均可在这4种表达菌中表达,但以BL21-CodonPlus(DE3)-RP的表达效果最好,可表达出可溶性并且产量较高的目的蛋白。免疫印迹结果表明,表达的目的蛋白可以被猪瘟阳性血清和针对E2蛋白的单克隆抗体所识别。结论只表达目的基因的抗原结构域,可以缩短表达片段的长度,有利于获得可溶性目的蛋白,并且具有良好的血清学反应的特异性。
[Objective] The E2 gene of Classical swine fever virus was expressed in prokaryotic expression system to obtain soluble fusion protein. [Method] Four antigen regions of E2 gene were amplified from recombinant plasmid S21 by the Polymerase Chain Reaction （PCR）. The four amplified fragments were cloned into pMAL-P2X vector, respectively. It was identified that the position and the size and the reading frame of the four recombinant vectors were all correct by RCR, restriction digestion and the sequence analysis. Then the four recombinant vectors were transducted into four expression germs and 16 strains of germs were obtained. [Result] SDS-PAGE indicated that these germs could express antigen region of E2 gene of CSFV and Western-blot indicated that these objective proteins could be recognized by the positive serum of CSFV and monoclonal antibody which is to E2 gene of CSFV. [Conclusion ] The antigen regions of the aimed gene were selected to be expressed, which could benefit the expression of soluble fusion proteins, and the speciality of serological reaction is satisfied.
出处
《中国农业科学》
CAS
CSCD
北大核心
2006年第4期814-818,共5页
Scientia Agricultura Sinica
基金
国家自然科学基金(30270984)
国家科技部"猪瘟检测技术平台(2004BA519A19-加强)"
关键词
猪瘟病毒
E2基因
抗原结构域原核表达
Classical swine fever virus
Antigen region of E2 gene
Construction and expression
