副溶血弧菌ZJ2003株两种铁调外膜蛋白的克隆、表达和免疫原性

Cloning,expression and immunogenicity analysis of two iron-regulated outer membrane proteins of Vibrio parahaemolyticus ZJ2003

从浙江省象山港网箱养殖大黄鱼病鱼分离的一株副溶血弧菌(Vibrio parahaemolyticus)ZJ2003中克隆了两种铁调外膜蛋白psuA和pvuA的基因(GenBank登录号:DQ141607、DQ141608),经PCR的方法去除两基因的信号肽编码序列后亚克隆到原核表达载体pET30a(+)中,经IPTG诱导获得大量表达。表达的重组蛋白以包涵体形式存在。包涵体蛋白经尿素法纯化及梯度透析法复性后以100μg/尾的剂量免疫大黄鱼,4周后经活菌攻毒获得80%的免疫保护率。免疫印迹分析表明,人工感染存活鱼的血清可以识别此两种重组蛋白。研究结果显示该两种铁调外膜蛋白具有良好的免疫原性,有可能作为高效疫苗成份。

In the coastal provinces of eastern China, V. parahaemolyticus was one of the causative agents of vibriosis that endangered the aquaculture of large yellow croaker （Pseudosciaena crocea ）, and no effective therapy was found. To throw some light for vaccine development against this pathogen, more information on protective antigens of the bacteria must be explored. Outer membrane proteins have been proved to be strong immunogenic in many gram-negative bacteria, however, little is known about this pathogen. In this study, genes encoding two iron-regulated outer membrane proteins of Vibrio parahaemolyticus ZJ2003, which was isolated from diseased large yellow croaker net-cage cultured in Xiangshan Bay, Zhejiang Province, were cloned and inserted into prokaryotic expression vector pET30a（ ＋ ）. The recombinant vectors were transfered into Escherichia colt BL21 （DE3） and expression of the recombinant proteins was induced by addition of 1 mmol/L IPTG to the exponential phase culture and continued shaking the bacteria for 6 h at 37 ℃. The recombinant proteins were over expressed in insolvable fusion bodies and were preliminary purified by urea method. Fourty large yellow croaker were individually immunized with 100/μg of the mixture of the recombinant proteins by intraperitoneal injection,while the positive control group were injected with 0.2 mL of formalin killed cells of the bacteria with the concentration of 10^9 cfu/mL,and the negative control was treated with the same volume of sterile PBS. Four weeks after vaccination, ten fish in each group were artificially challenged by intraperitoneal injection with live bacteria and the mortality was recorded in the following 14 d. Relative percent survival rate of the tested group reached 80%. Immunoblotting results showed that both of the recombinant proteins were recognized by antisera from survival fish. It is suggested that the recombinant proteins were immunogenic, and the two iron-regulated outer membrane proteins were expressed during in vivo infection and elicited protective immunity in the fish. PvuA and psuA are important IROMPs of V. parahaernolyticus, which acts as outer membrane receptor for the native siderophore vibrioferrin and a siderophore of unknown origin, respectively. Both of these proteins are essential elements of iron uptake systems of the bacteria, mutation of either of the genes resulted with impairment of the bacteria growth. Antibody against the IROMPs may block the transportation of siderophore iron complex and thus damage the survival and propagation of the bacteria. Since strong immunogenicity has been demonstrated by our study, these proteins should be given enough attention for their potential of vaccine components. [Journal of Fishery Sciences of China,2007,14（4） ：563 - 569]