布鲁杆菌BP26重组卡介苗的构建及对小鼠CD4＋和CD8＋T细胞的影响

Construction of a recombinant BCG secreting BP26 and the effects of BP26 on CD4＋ and CD8＋ T cells in mice

目的构建预防布鲁杆菌病的新型疫苗——布鲁杆菌BP26重组卡介苗（rBCG—BP26），观察rBCG—BP26对免疫小鼠CD4＋．CD8＋T细胞的影响。方法利用常规分子生物学技术构建重组穿梭分泌载体pMV261-Ag85B—BP26，电转入卡介苗（BCG）后经过卡那霉素抗性筛选，对获得的基因重组株进行PCR鉴定。采用Western免疫印迹法检测菌体和液体培养基中BP26蛋白表达情况。选用45只BALB／c小鼠进行安全性实验分析，将其分成3组：目标实验（rBCG—BP26）组、阳性对照（BCG）组、阴性对照（PBS）组，每组15只。分别在小鼠皮内接种100μl rBCG—BP26[含106克隆形成单位（CFU）]、BCG、PBS。观察各组小鼠体征，在小鼠免疫后的第10、20、30、40天称量体质量，并于尾部采血，用流式细胞仪分析CD4＋,CD8＋T细胞含量。结果成功构建rBCG—BP26重组疫苗株。在菌体和液体培养基中均能检测到BP26蛋白的表达。安全性实验分析表明，3组小鼠在免疫后第10、20、30、40天体质量比较，差异无统计学意义[rBCG—BP26组分别为（19．16±0．55）、（20．89±O．20）、（22．15±0．76）、（24．60±0．64）g；BCG组分别为（19．90±0．02）、（21．53±1．57）、（21．95±0．55）、（24．70±0．39）g；PBS组分别为（19．24±0．54）、（21．37±0．66）、（22．83±0．62）、（25．06±0．37）g；F值分别为2．468、0．331、1．520、0．739，P均〉0．05]。在免疫后第10、20天时，rBCG—BP26组小鼠全血CD4^＋T细胞含量（13．40％、26．70％）低于BCG组（26．70％、33．07％）和PBS组（33．85％、29.33％），rBCG—BP26组CD4＋／CD8＋T细胞含量比值随时间延长而逐渐增长（0．69％、1．27％、1．57％、1．70％）。结论rBCG-BP26能高效表达布鲁杆菌BP26蛋白，并具有毒力弱、能激活机体CD4＋、CD8＋T细胞的特点，可作为预防布鲁杆菌病的疫苗候选株之一。

Objective To develop a BP26 recombinant BCG （rBCG-BP26） vaccine, and to observe the effects of rBCG-BP26 on CD4＋, CD8＋ T ceils in immunized mice. Methods The recombinant shuttle vector pMV261-Ag85B-BP26 was constructed by using traditional molecular biological technology. The recombinant strains were obtained by kanamycin resistance screening and PCR identification after eleetroporation. Western blotting was used to detect the expression of recombinant BP26 vaccine in immunized mice. Safety experiment was carried out in three different groups： the target experiment （rBCG-BP26） group, the positive control （BCG） group and the negative control（PBS） group, 15 BALB/c mice in each group. Intradermal inoculations of 100 txl rBCG-BP26 [containing 106 colony forming units（CFU）], BCG, and PBS were carried out, respectively. Signs of mice in each group were observed. After immunization for 10, 20, 30, and 40 days, body weight was weighed, and tail blood was collected to observe the change of peripheral blood CD4＋ and CD8＋ T cells by flow cytometry. Results The rBCG-BP26 was successfully constructed. The expression of BP26 protein was detected in the liquid medium and the bacteria cells. The results of safety test analysis showed that there were no significant differences in signs and body weights （F = 2.468, 0.331, 1.520, 0.739, all P 〉 0.05）, between PBS group [ （19.24 ± 0.54）, （21.37 ± 0.66）, （22.83 ± 0.62）, （25.06 ± 0.37）g], BCG group[（19.90 ± 0.02）, （21.53 ± 1.57）, （21.95 ± 0.55）, （24.70 ± 0.39）g] and rBCG-BP26 group[ （19.16± 0,55）, （20.89 ± 0.20）, （22.15 ± 0.76）, （24.60 ± 0.64）g]. The results of flow eytometry showed that the percentages of CD4＋ T cell level were lower in BCG group（26,70%, 33.07%） and rBCG- BP26 group（13.40%, 26.70%） than that of the PBS group（33.85%, 29.33%） and the values of CD4＋/CD8＋ T cells increased in rBCG-BP26 group （0.69%, 1.27%, 1.57%, 1.70%） 10, 20 and 30 days after immunization. Conclusions Recombinant BCG-BP26 vaccine strain can express brucella BP26 protein efficiently. Furthermore, its virulence is mild, and it can activate CD4＋, CD8＋ T cells in the body. It can be used as one of eandidate vaccine strain against brucellosis.