摘要
以鸡传染性法氏囊病超强毒Gx株基因组RNA为模板,通过RT-PCR扩增VP1基因,经EcoRⅠ和XhoⅠ双酶切处理后与经相同酶切处理的pET-30a原核表达载体连接,将酶切、PCR及测序鉴定正确的阳性重组子转化BL21(DE3)感受态细胞,融合蛋白His-VP1经IPTG诱导表达后主要以包涵体形式存在。Western blot检测到约97ku的目的蛋白能够与特异性的VP1单克隆抗体反应。将纯化的目的蛋白免疫新西兰白兔后获得了抗VP1蛋白的血清。该血清能够与重组杆状病毒rBacGxVP1感染的sf9细胞发生特异性反应,而且其ELISA抗体效价达到1∶25600。鸡传染性法氏囊病超强毒VP1基因的表达与兔抗血清的制备为病毒的检测及VP1功能的研究提供了有效工具。
VP1 gene of very virulent infectious bursal disease virus Gx strain was amplified and cloned into prokaryotic expression vector pET-30a based on restriction enzyme analysis. After sequencing,the recombinant expression vector pET-30aGxVP1 was transformed into the competent BL21 (DE3). The fusion protein His-VP1 was expressed after induced by IPTG and presented mainly in inclusion body. Western blot analysis showed that the fusion protein His-VP1 could react with antibody against VP1 specifically. The specific antiserum against VPI was produced in rabbit immunized by the purified His-VP1. The positive signal was detected in sf9 cell infected with the recombinant baculovirus rBacGxVP1 by indirect immunofluorescence assay. Besides,the antibody titer was 1:25 600 with EUSA. The production of VP1 protein and antiserum will be a useful tool for the virus detection and study on VP1.
出处
《中国家禽》
北大核心
2011年第2期11-14,共4页
China Poultry
基金
现代农业肉鸡产业技术体系建设(nycytx-42-G3-01)
关键词
鸡传染性法氏囊病超强毒
VP1
原核表达
兔抗VP1血清
very virulent infectious bursal disease virus
VP1
prokaryotic expression
rabbit antiserum against VP1