重组卡介苗-BE1726D的鉴定及其免疫原性评价

Identification of immunogenicity evaluation of recombinant BCG-BE1726D

目的鉴定重组卡介苗-BE1726D(rBCG-BE1726D)特异性蛋白的表达,并评价其在小鼠体内的免疫原性。方法将冻干的菌株BCG-SSI和rBCG-BE1726D扩大培养后,超滤浓缩收集上清,超声破碎离心后收获菌体,Western blot法进行特异性蛋白检测。用rBCG-BE1726D和BCG-SSI菌株经腹股沟皮下免疫BALB/c小鼠6周后,分离小鼠脾淋巴细胞,ELISPOT及ELISA法检测脾淋巴细胞干扰素γ(interferonγ,IFNγ)的分泌水平;流式细胞术检测小鼠脾淋巴细胞CD4^+和CD8^+ T细胞的比例。结果菌株rBCG-BE1726D中存在Ag85B、ESAT-6、RV2626c蛋白;菌株BCGSSI中仅存在Ag85B蛋白。经Ag85B刺激后,BCG-SSI组和rBCG-BE1726D组产生的细胞斑点数均明显高于PBST对照组(P<0.05);经PPD刺激后,rBCG-BE1726D组产生细胞斑点数量显著高于BCG-SSI组和PBST对照组(P<0.000 1);各组间ESAT-6、Rv2626c、Rv1733c、RpfD刺激所产生的细胞斑点数差异无统计学意义(P>0.05)。经Ag85B刺激后,BCG-SSI组和rBCG-BE1726D组产生IFNγ的水平显著高于PBST对照组(P<0.05);经PPD刺激后,rBCG-BE1726D组的IFNγ分泌水平与BCG-SSI组差异无统计学意义(P>0.05);各组间ESAT-6、Rv1733c、Rv2626c、RpfD刺激产生的IFNγ分泌水平差异无统计学意义(P>0.05)。rBCG-BE1726D组CD4^+、CD8^+细胞总比例及CD4^+ T/CD8^+ T比值明显低于BCG-SSI组和PBST对照组(P<0.05),CD8^+ T细胞比例明显高于BCG-SSI组和PBST对照组(P<0.05)。结论 rBCG-BE1726D有较强的免疫原性,可明显提高BALB/c小鼠的免疫应答水平,但与BCG-SSI比较,其免疫原性无明显优势,需对疫苗的保护力进行进一步研究。

Objective To identify the expression of specific recombinant BCG-BE1726D protein （rBCG-BE1726D） and evaluate its immunogenicity in mice. Methods Freeze-dried BCG-SSI and rBCG-BE1726D strains were subjected to expand cultivation of which the supernatant was collected by uhrafiltration concentration, while bacteria by uhrasoniction and centrifugation, and determined for specific proteins by Western blot. BALB/c mice were immunized s.c. with rBCG- BE1726D and BCG strains, of which splenic lymphocytes were isolated 6 weeks later, then determined for the secretion level of interferon γ （IFNγ） by ELISPOT and ELISA, and for percentages of CD4＋ amd CD8＋ T lymphocytes by flow cytometry. Results Ag85B, ESAT-6 and RV2626c proteins were detected in rBCG-BE1726D strain, while only Ag85B in BCG-SSI. After stimulation with Ag85B, the number of cell spots in BCG-SSI and rBCG-BE1726D groups were significantly higher than those in PBST control group （P 〈 0. 05）. However, after stimulation with PPD, the number of cell spots in rBCG-BE1726D group was significantly higher than those in BCG-SSI and PBST control groups （P 〈 0. 000 1 ）. No significant differences were observed in the numbers of cell spots produced by stimulation with ESAT-6, Rv2626c, Rv1733c and RpfD in various groups （P 〉 0. 05）. After stimulation with Ag85B, the secretion of IFNγ in BCG-SSI and rBCG-BE1726D groups were significantly higher than that in PBST control group （P 〈 0. 05）. However, after stimulation with PPD, the secretion level of IFNγ in rBCG-BE1726D group showed no significance with that in BCG-SSI group （P 〉 0. 05）. The secretion levels of IFNγ after stimulation with ESAT-6, Rv2626c, Rv1733c and RpfD in various groups showed no significant difference （P 〉 0. 05）. The total percentage of CD4＋ and CD8＋ cells as well as ratio of CD4＋ to CD8＋ Tlymphocytes in rBCG-BE1726D group were significantly lower, while the percentage of CD8 ＋ T lymphocytes was significantly higher, than those in BCG-SSI and PBST control groups （P 〈 0. 05）. Conclusion Recombinant BCG- BE1726D showed high immunogenicity, which enhanced the immune response level of BALB / c mice significantly. However, the immunogenicity of rBCG-BE1726D showed no obvious advantages as compared with BCG-SSI, so the protection of this vaccine should be further studied.