Zhou Qi, Gu Xiangxue, Xu Zejun, Huang Rong, Huang Junsheng, Li Haoran, Li Tao, Zhou Xiaoya, Su Guangzhi, Yin Dongdong, Wang Guijun
College of Animal Science and Technology, Anhui Agricultural University, Anhui province key laboratory of veterinary pathobiology and disease control, Hefei 230036, China.
College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2018 Nov;34(11):1036-1040.
Objective To obtain the envelope protein B2L of orf virus(ORFV) and prepare highly specific polyclonal antibody against B2L. Methods The B2L gene was amplified by PCR, and subcloned into the vectors pET-32a and p3×FLAG-CMV-14, pET-32a-B2L followed by expression of B2L protein in E.coli, and induction of the target protein by IPTG, purification by urea solution and identification via Western blot analysis. Furthermore, the BALB/c mice were immunized with B2L protein to prepare highly-specific anti-B2L polyclonal antibody. In addition, plasmid p3×FLAG-B2L was transfected into Vero cells and BHK-21 cells which was used to confirm its specificity and antigenicity by indirect immunofluorescence assay(IFA). Results Western blotting demonstrated that the B2L protein had high-level specificity. The results of IFA indicated that the anti-B2L specific polyclonal antibody had specificity and reactivity. Then Immunohistochemistry showed that it could neutralize natural ORFV. Conclusion Highly specific and purified polyclonal antibody against B2L protein of the ORFV was successfully prepared.
目的 获得羊口疮病毒（ORFV）的包膜蛋白B2L，并制备抗B2L的高特异性多克隆抗体。方法 通过PCR扩增B2L基因，并亚克隆至载体pET-32a和p3×FLAG-CMV-14中，pET-32a-B2L在大肠杆菌中表达B2L蛋白，经IPTG诱导靶蛋白表达，用尿素溶液纯化，通过Western blot分析进行鉴定。此外，用B2L蛋白免疫BALB/c小鼠制备高特异性抗B2L多克隆抗体。另外，将质粒p3×FLAG-B2L转染至Vero细胞和BHK-21细胞，通过间接免疫荧光试验（IFA）确认其特异性和抗原性。结果 Western blotting显示B2L蛋白具有高度特异性。IFA结果表明抗B2L特异性多克隆抗体具有特异性和反应性。免疫组化显示其可中和天然ORFV。结论 成功制备了抗ORFV B2L蛋白的高特异性纯化多克隆抗体。
