摘要
用疑似患猪流行性腹泻病(PED)的病猪肠病料,根据猪流行性腹泻病毒(PEDV)S糖蛋白基因设计引物进行RT-PCR扩增,获得531 bp的PEDV部分保护性抗原基因,将其克隆入pMD18-T载体后测序,核苷酸序列与PEDV CV777株相应序列的同源性为99.4%。根据测序结果和表达载体特点,设计一对引物,扩增PEDV部分保护性抗原基因(COE基因)501 bp片段。将COE基因与乳酸乳球菌表面表达载体pNZ8149进行连接,电击转化入食品级乳酸乳球菌NZ3900细胞。重组菌以1 ng/mL乳链菌肽(Nisin)诱导,通过SDS-PAGE和Western blot分析,PEDV部分S蛋白成功表达,并具有反应原性。间接免疫荧光试验表明,重组菌表达蛋白定位于菌体细胞表面。
The partial S glycoprotein gene of porcine epidemic diarrhea virus （PEDV） was amplified by RT-PCR and sequenced. The gene consisted of 531 bp and shared 99.4% nueleotide homology with PEDV CV777 strain （AF353511） published on GenBank. The COE gene （about 501 bp） in S glycoprotein gene was subcloned into the Lactococcus lactis vectors pNZ8149 and transformed into Lactococcus lactis NZ3900 by electroporation. The recombinant protein was detected by Western blot and IFA experiments after the bacteria were induced by 1 ng/mL nisin. The results indicated that the molecular weight of the expressed recombinant protein was about 20 kDa; The protein had the reactionogenicity with antibody against PEDV as expected; The protein was secreted and located on the surface of the bacteria.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2008年第12期1743-1747,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金资助项目(30571382)
