结核分枝杆菌Rv1009结构域多肽的免疫学特性研究

Immunological properties of Rv1009 domain from Mycobacterium tuberculosis

目的研究结核分枝杆菌Rv1009结构域多肽的免疫学特性。方法用原核表达的Rv1009结构域多肽免疫BALB／c小鼠3次，每次间隔2周。用ELISA法检测免疫小鼠血清中特异性抗体滴度。分离免疫小鼠的脾淋巴细胞，体外用抗原再刺激后，用MTF比色法检测免疫小鼠脾淋巴细胞的增殖指数。ELISA方法检测淋巴细胞悬液中γ干扰素（IFN-γ）、白细胞介素（IL）-10和IL-12的产生水平。另一部分免疫的小鼠经尾静脉感染MTB毒株H37Rv，4周后，计数脾脏细菌负荷数。结果Rv1009结构域多肽免疫小鼠血清特异性抗体滴度为1：12800。淋巴细胞增殖指数为2．40±0．18，明显高于生理盐水对照组的0．90±0．21。ELISA方法检测Rvl009结构域多肽免疫组IFN-γ IL—10和IL-12水平为（1432±30）ng／L、（503±11）ng／L和（311±11）ng／L，显著高于生理盐水对照组的（256±20）ng／L、（76±6）ng／L和（56±8）ng／L（P〈0．01）。与生理盐水免疫组（细菌负荷6．64±0．13）相比较，Rv1009结构域多肽免疫组小鼠，对攻击感染后抗MTB在脾脏中增殖有显著作用（细菌负荷为4．86±0．14，P〈0．05），但不及BCG免疫组的3．81±0．16。结论Rv1009结构域多肽有可能作为新型结核疫苗的候选组分。

Objective To investigate the immunological properties of Rvl009 domain. Methods BALB/c mice were immunized with Rv1009 domain three times at 2-week interval. ELISA was used to detect the anti-Rv1009 domain antibody titer in the sera of immunized mice sera. The spleen lymphocytes of the immunized mice were separated and the stimulation index （SI） was measured by MTT eolorimetry. Levels of secreted IFN-γ , IL-IO and IL-12 upon specific antigen stimulation were detected by ELISA. The BALB/c mice immunized with Rv1009 domain were intravenously infected with MTB H37 Rv. Four weeks after the final injection, the number of CFU in spleens was determined. Results The titer of the specific antibody in sera of the immunized BALB/c mice was 1：12 800. The SI of Rvl009 domain immunized group （2.40 ±0. 18） was significantly higher than that of saline immunized group （0.90 ±0. 21 ）. The IFN-γ,IL-10 and IL-12 levels in culture supernatant of spleen lymphocytes from the fusion proteins immunized mice was （ 1 432 ± 30） ng/ L, （503 ± 11 ） ng/L and （311 ± 11 ） ng/L respectively, significant different from that of saline immunized group [ （ 256 ± 20 ） ng/L, （ 76± 6 ） ng/L and （ 56 ± 8 ） ng/L, P 〈 0. 01 ]. Four weeks after the final injection, compared with normal saline immunized mice （6.64 ± 0. 13 ）, dramatic reduction in MTB replication was observed in the spleen （4. 86 ± 0. 14 ） from BALB/c mice immunized with fusion proteins following a subsequent MTB H37 Rv challenge, but the protection efficacy of mice immunized with Rv1009 domain was not as good as that of BCG vaccination group （3.81 ± 0. 16）. Conclusion Rv1009 domain can be used as a candidate for the new TB vaccine.