表面展示新城疫病毒部分F蛋白的重组干酪乳杆菌的构建及其免疫原性

Immunogenicity of the truncated NDV F protein surface-displayed on Lactobacillus casei

摘要:

本研究旨在构建重组干酪乳杆菌pLA-Newcastle disease virus (NDV)-F/Lactobacillus casei，获得表达产物，并探讨其免疫效果。利用PCR扩增携带部分主要抗原表位的NDV F基因，与穿梭质粒pLA连接转化至大肠杆菌BL21 (DE3) 中，筛选阳性重组质粒，将其电转化至干酪乳杆菌中，构建重组干酪乳杆菌pLA-NDV-F/L. casei，应用PCR鉴定阳性菌株，Western blotting鉴定重组菌反应原性，间接免疫荧光、流式细胞术和激光共聚焦检测蛋白表达情况。试验选用14日龄雏鸡，各组免疫方式为口服+滴鼻。设立pLA-NDV-F/L. casei两次免疫组和三次免疫组、弱毒疫苗组、pLA/L.casei、未攻毒PBS组和攻毒PBS组。间接ELISA方法检测雏鸡血清IgG、肠道、鼻腔、肺脏中sIgA抗体效价，评价试验组雏鸡攻毒保护率。结果表明，有94.10%的重组菌表达了F蛋白，且高效表达在干酪乳杆菌细胞表面，蛋白大小为62 kDa，并能与抗NDV阳性血清特异性结合。各免疫组anti-F IgG和sIgA抗体水平显著高于对照组，pLA-NDV-F/L. casei三次免疫组抗体持续时间比两次免疫组延长28 d，抗体峰值没有显著差异。免疫pLA-NDV-F/L. casei三次、两次、弱毒疫苗、pLA/L. casei和PBS的攻击保护率分别为80%、80%、90%、0%和0%。因此，利用干酪乳杆菌表达体系成功表达了携带部分抗原表位的NDV F基因，具备良好的反应原性和免疫原性，可诱导机体产生保护性免疫应答。

Abstract:

To evaluate immune efficacy of the recombinant Lactobacillus casei, we constructed pLA-Newcastle disease virus (NDV)-F/L. casei and obtained the expression products. PCR amplified the NDV F gene carrying part of the major epitopes. The target gene was inserted to the shuttle plasmid pLA, and then transformed into Escherichia coli BL21 (DE3) in order to screen positive recombinant plasmid. The positive recombinant plasmid was transformed into L. casei by electroporation to construct pLA-NDV-F/L. casei. The positive strains were identified by PCR. The reactivity of the recombinant bacteria was identified by Western blotting and the protein expression was detected by indirect immunofluorescence, flow cytometry and laser confocal microscopy. The 14-day-old chickens in each group were vaccinated by oral plus nose drops. The pLA-NDV-F/L. casei twice immunization group and three times immunization group, the commercial vaccine group, the pLA/L. casei group, the unchallenge PBS and the challenge PBS group were established. IgG in serum and sIgA in the lavage fluid of intestinal, nasal and lung were detected by ELISA. The protection rate of chickens was evaluated. The results showed that 94.10% of the recombinant bacteria expressed the F protein. The recombinant protein was highly expressed on the surface of L. casei with a protein size of 62 kDa, which specifically bound to anti-NDV serum. The levels of anti-F IgG and sIgA antibodies in each test group were significantly higher than those in the control groups. The duration of antibody in the pLA-NDV-F/L. casei three-time immunization group lasted 28 days longer than that in the twice immunized group, and there was no significant difference between antibody peak values. The attack protection rates in each group of immunized pLA-NDV-F/L. casei three times, twice, attenuated vaccine, pLA/L. casei and PBS were 80%, 80%, 90%, 0% and 0%, respectively. Therefore, the antigenic protein of NDV F was successfully expressed by L. casei expression system, which has of reactogenicity and immunogenicity, and could induce protective immune responses in chickens.