鸡传染性法氏囊病超强毒VP1基因的原核表达及兔抗血清的制备

Prokaryotic Expression of VP1 Gene of Very Virulent Infectious Bursal Disease Virus and Production of Antiserum against VP1 in Rabbit

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.