表达空肠弯曲菌CfrA蛋白重组乳酸乳球菌的构建及其免疫效果

Immunological modulation of chickens by recombinant Lactococcus lactis expressing ferric enterobactin receptor CfrA of Campylobacter jejuni

摘要:

[目的] 本试验将空肠弯曲菌肠菌素受体蛋白CfrA编码基因导入食品级乳酸乳球菌表达系统，然后将重组乳酸乳球菌口服免疫鸡，降低空肠弯曲菌在鸡肠道中的定殖。[方法] 利用PCR分别扩增空肠弯曲菌cfrA全基因及其N端片段，插入食品级表达载体pNZ8149多克隆位点并转化乳酸乳球菌NZ3900，通过Western blot鉴定重组菌株CfrA蛋白表达情况，同时通过筛选nisin浓度、温度、时间等诱导条件优化重组蛋白表达水平；进而将重组乳酸乳球菌经口服免疫SPF鸡，免疫后分别测定乳酸乳球菌自鸡体内的排出情况、以及诱导CfrA血清抗体和粘膜抗体水平，最后将空肠弯曲菌口服攻毒免疫后的鸡，通过测定鸡泄殖腔棉拭子中空肠弯曲菌的数目来判定口服免疫效果。[结果] Western blot检测显示CfrA全基因及其N端片段均可在重组乳酸乳球菌胞内可溶性表达，不分泌，筛选的最佳诱导表达条件为nisin浓度25 ng/mL、温度37℃、时间1 h。口服乳酸乳球菌10 d内自鸡体完全排空；鸡口服免疫后可产生CfrA蛋白特异性的血清IgG和肠粘膜sIgA抗体；重组乳酸乳球菌口服免疫后空肠弯曲菌在鸡体内的增殖速度显著低于对照组。[结论] 成功构建了重组CfrA蛋白的食品级乳酸乳球菌诱导表达系统；表达CfrA蛋白的重组乳酸乳球菌口服免疫鸡对空肠弯曲菌在鸡肠道的定殖具有一定的抑制作用，为研制重组乳酸菌口服家禽免疫制剂防治空肠弯曲菌奠定了基础。

Abstract:

[Objective] The purpose of this study is to reduce the colonization level of Campylobacter jejuni in chicken intestine by oral immunization of recombinant Lactococcus lactis expressing the ferric enterobactin receptor CfrA of C. jejuni.[Methods] The whole cfrA gene and its N-terminal fragments were amplified by PCR, inserted into the expression vector pNZ8149 and transformed into L. lactis NZ3900. Based on the expression of CfrA in recombinant L. lactis by Western blot, the expression level was optimized by screening nisin concentration, induction temperature and time. Then the recombinant L. lactis strains were used to orally immunize specific-pathogen-free chickens. After oral immunization, the duration of recombinant L. lactis in chickens was determined by PCR, and the antibody levels of anti-CfrA serum IgG and intestinal mucosal sIgA were measured by ELISA. Finally, the immunized chickens were orally inoculated with C. jejuni to evaluate the inhibitory effect of recombinant L. lactis on colonization of C. jejuni.[Results] Western blot results determined that the whole cfrA gene and its N-terminal fragments were both expressed in recombinant L. lactis in soluble forms whereas no secreted CfrA protein was detected outside bacterial cells. The optimal conditions for inducing the expression were grown at 37℃ for 1 h with nisin concentration of 25 ng/mL. Detection of chicken cloacal swabs showed that the duration of oral L. lactis was less than 10 days in chicken. The immunized groups produced higher antibody titers of anti-CfrA specific serum IgG and mucosal sIgA than the control groups. Moreover, the colonization rate of C. jejuni in the immunized groups was significantly lower than that in the control groups.[Conclusion] Oral immunization of chickens with recombinant L. lactis expressing CfrA inhibited the colonization of C. jejuni. Our findings can be useful to develop oral vaccines with recombinant Lactobacillus for control of C. jejuni infection in chickens.