重组Bb(pGEX-OprF-I)疫苗的构建及对小鼠的诱导保护作用

Construction of recombinant Bb (pGEX-OprF-I) vaccine of Pseudomonas aeruginosa and its protection elicited in mice

目的构建和鉴定重组两歧双歧杆菌介导的铜绿假单胞菌外膜蛋白F-I[rBb(pGEX-OprF-I)]疫苗,研究其对小鼠抗铜绿假单胞菌感染的免疫保护作用。方法 PCR扩增OprF和OprI基因,采用基因拼接法剪切,得到OprF-I融合基因,双酶切后定向克隆至载体pGEX-1λT,构建重组质粒pGEX-OprF-I,将该质粒电转化两歧双歧杆菌(Bb),构建rBb(pGEX-OprF-I)疫苗并进行双酶切和PCR鉴定,经异丙基-β-D硫代-吡喃半乳糖苷(IPTG)诱导表达, SDS-PAGE和Western blot法分析和鉴定表达产物。将21只BALB/c鼠随机分成疫苗组、 Bb-pGEX-1λT空载体和Bb对照组,取5×10~8菌落形成单位(CFU)疫苗口服灌胃,每周连续接种3 d,持续3周。初次免疫后4周用5×10~7 CFU PA01株滴鼻攻击,攻击后2周无菌杀鼠,计数肺组织的细菌菌落数。常规ELISA检测免疫前、初次免疫后4周和攻击后2周的小鼠静脉血中IgG水平。结果 PCR扩增出1289 bp的OprF-I融合基因;双酶切和PCR鉴定证实该基因插入pGEX-1λT并转化Bb,成功构建rBb (pGEX-OprF-I)疫苗;SDS-PAGE显示该疫苗经IPTG诱导可表达相对分子质量(M_r)约68 000的融合蛋白;Western blot表明该蛋白能被铜绿假单胞菌感染的鼠血清特异性识别。疫苗组的肺组织细菌菌落数较对照组明显减少;免疫小鼠血清IgG水平在初次免疫后4周和攻击后2周依次升高,且高于同一时间点的其余对照组。结论成功构建了铜绿假单胞菌rBb(pGEX-OprF-I)疫苗,该疫苗在小鼠抗铜绿假单胞菌感染过程中可产生一定的免疫保护作用。

Objective To construct and identify Bifidobacterium bifidum-vectored outer membrane protein F-I[rBb(pGEX-OprF-I)] vaccine of Pseudomonas aeruginosa and observe its protection against Pseudomonas aeruginosa infection in mice. Methods OprF and OprI genes were amplified by PCR, then the OprF-I fusion gene obtained by gene SOEing was digested and ligated into the vector pGEX-1λT to construct the recombinant plasmid pGEX-OprF-I. The plasmid was transformed into Bifidobacterium bifidum(Bb) by electroporation, and the rBb(pGEX-OprF-I) vaccine was constructed and identified by double enzyme digestion and PCR. Expression products of the vaccine induced by IPTG were analyzed and identified by SDS-PAGE and Western blot analysis. Twenty-one BALB/c mice were randomly divided into rBb(pGEX-OprF-I) vaccine group, Bb-pGEX-1λT empty vector group and Bb control group. The 5×10~8 CFUs vaccine was intragastrically administered for 3 consecutive days per week for 3 weeks. All mice were challenged intranasally with 5×10~7 CFUs of PA01 strain at the 4th week after the first immunization. At the 2nd week after the challenge, all mice were sacrificed to count the lung bacteria loads. IgG levels in sera from the mice before immunization, 4th week after the first immunization and 2nd week after the challenge were detected by routine ELISA. Results A total of 1289 bp OprF-I fusion gene was amplified by PCR. Double enzyme digestion and PCR identification confirmed that the gene was ligated into pGEX-1λT and transformed into Bb, and the rBb(pGEX-OprF-I) vaccine was successfully constructed. SDS-PAGE showed that the fusion protein with a relative molecular mass(M_r) of about 68 000 could be expressed by IPTG-induced vaccine. Western blot analysis indicated that the protein could be specifically recognized by the sera of Pseudomonas aeruginosa-infected mice. The number of bacteria colonies in the lung of the mice immunized with rBb(pGEX-OprF-I) vaccine was significantly lower than that of the control group. The IgG levels in the sera of the immunized mice increased successively at 4th week after the first immunization and 2nd week after the challenge, and higher than that in the other control groups at the same time point. Conclusion The rBb(pGEX-OprF-I) vaccine has been successfully constructed, and it may take a certain protective effect on the mice against Pseudomonas aeruginosa infection.