重组两歧双歧杆菌疫苗的构建、鉴定、表达及其免疫保护力研究  

Construction, identification and expression of recombinant Bifidobacterium bifidum vaccine of Pseudomonas aeruginosa and its protective effect

作　　者：江帆 李文桂[1] 刘潇[1] Jiang Fan;Li Wengui;Liu Xiao(Institute of Infectious and Parasitic Diseases,the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院传染病寄生虫病研究所,400016

出　　处：《中华地方病学杂志》2019年第1期31-35,共5页Chinese Journal of Endemiology

基　　金：重庆市科委地方病重大专项基金(2008AB5055、2008AB5008、2008AB5054).

摘　　要：目的 构建铜绿假单胞菌重组(r)两歧双歧杆菌(Bb)疫苗,研究疫苗免疫后铜绿假单胞菌攻击小鼠的免疫保护力。 方法 将PCR扩增的铜绿假单胞菌重组外膜蛋白H(OprH)基因克隆至载体pGEX-1λT,电穿孔转化两歧双歧杆菌,得到rBb-pGEX-OprH疫苗。将疫苗进行PCR鉴定,再以异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达,表达产物用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析,并行蛋白质免疫印迹(Western blot)鉴定。将21只BALB/c小鼠按照随机数字表法分为rBb-pGEX-OprH疫苗组(简称疫苗组)、空载体组和Bb对照组(每组7只),分别用rBb-pGEX-OprH、rBb-pGEX-1λT或Bb行灌胃免疫,每天1次,连续3 d,持续3周,于首次免疫后4周以5 ×10^7菌簇形成单位(CFU)铜绿假单胞菌滴鼻攻击,每天1次,连续3 d。攻击2周后处死小鼠,计数肺细菌负荷。另于免疫前、首次免疫后4周、攻击感染后2周取静脉血,酶联免疫吸附试验(ELISA)法测定小鼠血清IgG水平。 结果 PCR扩增出660 bp的OprH基因;PCR鉴定显示,rBb-pGEX-OprH疫苗构建成功;SDS-PAGE证实,重组疫苗可表达相对分子质量为47 ×10^3的融合蛋白,与预期结果一致;Western blot结果显示,表达蛋白可被铜绿假单胞菌感染的小鼠血清识别。3组小鼠肺细菌菌落数比较差异有统计学意义(F = 1 506.793,P < 0.05),疫苗组肺细菌菌落数[(7.691 ± 0.069)lgCFU/g]低于空载体组[(8.855 ± 0.027)lgCFU/g]和Bb对照组[(8.958 ± 0.037)lgCFU/g,P均< 0.05]。疫苗免疫后4周,3组小鼠血清IgG水平比较差异有统计学意义(F = 77.216,P < 0.05),其中疫苗组血清IgG水平(0.078 ± 0.005)明显高于空载体组(0.054 ± 0.004)和Bb对照组(0.052 ± 0.004,P均< 0.05)。 结论 铜绿假单胞菌rBb-pGEX-OprH疫苗构建成功,且可诱导小鼠产生一定的保护力。Objective To construct a recombinant Bifidobacterium bifidum (Bb) vaccine of Pseudomonas aeruginosa, identify its protective effect in mice after immunization with the vaccine and challenged by Pseudomonas aeruginosa. Methods The PCR-amplified outer membrane protein OprH gene was cloned into vector pGEX-1λT to obtain the plasmid pGEX-OprH. The pGEX-OprH was then electroporated into Bb to obtain the rBb-pGEX-OprH vaccine. After PCR identification, the expression of rBb-pGEX-OprH was induced by Isopropyl β-D-Thiogalactoside (IPTG), and the expression products were analyzed by SDS-PAGE and characterized by Western blotting;21 BALB/c mice were subdivided by random number table into vaccine group, empty vector group and Bb group (7 mice in each group), and immunized by intragastrically administration with rBb-pGEX-OprH, rBb-pGEX-1λT or Bb, respectively. Mice were immunized once a day and three times a week for three consecutive weeks, then the mice were challenged intranasally with Pseudomonas aeruginosa at four weeks after the first immunization, once a day for three consecutive days. Two weeks after the challenge, the mice were sacrificed to detect their lung bacterial loads. Sera were collected before vaccination, four weeks after the first immunization, and two weeks after the challenge. The sera were analyzed by enzyme-linked immunosorbent assay (ELISA) to detect the level of IgG. Results OprH gene of 660 bp was amplified by PCR;PCR results showed that the rBb-pGEX-OprH was constructed successfully;SDS-PAGE confirmed that the recombinant vaccine expressed a relative molecular mass 47 ×10^ fusion protein, which was consistent with the expected result;Western blot results showed that the expressed protein could be identified by sera in mice infected with Pseudomonas aeruginosa. Differences of lung bacterial colonies between the three groups were statistically significant (F= 1 506.793, P < 0.05). The number of bacterial colonies in the vaccine group [(7.691 ± 0.069) lgCFU/g] was lower than that of the empty ve

关 键 词：铜绿假单胞菌 二裂菌属 双歧杆菌属 疫苗 

分 类 号：R392[医药卫生—免疫学]