家兔皮肤液化病理模型与分枝杆菌毒力差异相关性研究及结核整合蛋白疫苗研究

发布时间：2018-04-25 04:26

本文选题：结核分枝杆菌 + 病理　；参考：《兰州大学》2011年硕士论文

【摘要】：1.家兔皮肤液化病理模型与分枝杆菌毒力差异相关性研究目的：利用卡介苗BCG、结核分枝杆菌减毒株H37Ra、耻垢分枝杆菌Mycobacterium smegmatis(M. smegmatis)以及H37Ra互补株(分别重组了11种来源H37Rv野生型基因的H37Ra菌株)感染家兔皮肤,观察皮肤病理变化情况,可以快速有效地评价不同分枝杆菌毒力以及引起家兔皮肤液化能力的差异。方法：实验一中家兔随机分成6组,分别进行腹部两侧皮内注射5×106CFU的BCG、H37Ra和M. smegmatis的活菌液和热灭活菌液；距初次免疫后约42天,每组以相同剂量的同种菌液对家兔进行再次皮内免疫,观察家兔皮肤病理变化。实验二中家兔随机分成13组,设立H37Ra株和pOLYG载体组为对照组,实验组为H37Ra互补株组包括P10、P11、P12…P20组。以上菌株分别感染家兔皮肤,每组注射剂量分为三种即高剂量(5x107CFU)、中剂量(5×106CFU)、低剂量(5x105CFU)。六周后进行再次免疫,观察家兔皮肤损伤情况。结果：实验一,家兔分别经皮内接种BCG、H37Ra和M. smegmatis的活菌液和热灭活菌液后,活菌组家兔可以观察到皮肤有明显的炎症反应和脓肿形成以及液化、溃疡的发生；而热灭活组都不能引起家兔皮肤发生明显的病理变化。再次免疫后可以发现各组病理反应程度会加重。活菌组引起家兔皮肤病变的严重程度为：BCG引起的病变反应最强,其次是H37Ra, M. smegmatis的反应最弱。实验二,H37Ra互补株感染家兔皮肤以后,高剂量组(5x107CFU)和中剂量组(5×106CFU)均可诱导产生强烈的家兔皮肤病理改变。对于不同H37Ra互补株,所引起的病灶反应程度存在差异：H37Ra互补株P10(重组了H37Rv的基因pstAl)引起的病灶体积最大,其次是P19(lpdA)、P17(padA)、P15(mazG)、P18(nrdH)、P14(pknH) (*p=0.05 vs. H37Ra control)。然而,P11(phoP)、P12(marR family)、P13(luxR/uhpA family)、P16(nadD):和]P20(ilvD)与载体对照pOLYG和H37Ra引起的病灶反应比较相似。与其他组相比,P18(nrdH)和P14(pknH)能促使家兔皮肤病灶发生最强烈的液化反应；P10(pstAl)、P11(phoP)、P13(luxR/uhpA family)、P16(nadD)和P20(ilvD)组的液化强度与对照组pOLYG和H37Ra比较没有明显差异。结论：剂量(5×106 CFU)的活菌BCG、H37Ra和M.smegmatis能够引起家兔皮肤发生液化现象,其中BCG组病灶反应最强烈,其次是H37Ra组,最弱为M.smegmatis组。当分枝杆菌被灭活后,便丧失了引发液化反应的能力。而且利用这种家兔皮肤模型可以有效评价不同H37Ra互补株引起的病理变化的差异。 2.无标签结核融合蛋白ESAT6-Ag85B/TB10.4-Ag85B/ESAT6-TB8.4/ TB10.4-TB8.4的克隆构建和免疫原性检测目的：克隆构建无标签结核分枝杆菌融合蛋白ESAT6-Ag85B/TB10.4-Ag85B/ESAT6-TB8.4/TB10.4-TB8.4,并利用动物实验对此融合蛋白的免疫原性进行初步评价。方法：应用分子克隆技术设计含有不同酶切位点的ESAT6、TB10.4、TB8.4和Ag85B的引物,以H37Rv-DNA为模板PCR扩增出相应大小的基因片段,通过基因工程技术进行重组而获得重组质粒pET30a-ESAT6-Ag85B/pET30a-TB10.4-Ag85B/pET30a-ESAT6-TB8.4/pET30a-TB10.4-TB8.4。将重组质粒转化入大肠杆菌菌株BL21,进行IPTG诱导表达,采用IEX(离子交换色谱层析)和HIC(疏水作用色谱层析)两种方法进行融合蛋白的纯化。最后将蛋白与佐剂DDA混合配制成蛋白疫苗进行动物实验,将C57BL/6小鼠随机分成9组,设立PBS、BCG和单个抗原作为对照组,融合蛋白组为实验组。免疫后第14周采用ELISA技术检测免疫后小鼠的脾脏淋巴细胞分泌INF-y水平。结果：PCR扩增获得的ESAT6、TB10.4、TB8.4和Ag85B序列与GenBank中完全一致。融合蛋白在大肠杆菌中表达产物与预计大小相吻合,融合蛋白ESAT6-Ag85B分子量约38KD、融合蛋白TB10.4-Ag85B分子量约42KD,二者以包涵体的形式表达,最后应用离子交换层析和疏水层析可纯化此两种复性后的蛋白；融合蛋白ESAT6-TB8.4分子量约18KD、融合蛋白TB10.4-TB8.4分子量约19KD,二者以上清液表达为主,应用离子交换层析和疏水层析可纯化此两种可溶性蛋白。ELISA检测结果显示：免疫后小鼠脾脏淋巴细胞受单个蛋白ESAT6和TB8.4刺激以后,融合蛋白ESAT6-TB8.4组产生的IFN-y高于BCG组和ESAT6组(**p0.05 vs.BCG and ESAT6)。与BCG组、TB8.4组相比,当受单个蛋白TB8.4刺激以后,融合蛋白TB10.4-TB8.4组分泌IFN-y水平高于BCG组和TB8.4组(**p0.05 vs.BCG and TB8.4);而针对TB10.4特异性抗原表位刺激时,融合蛋白TB10.4-TB8.4组分泌IFN-γ水平高于BCG组(p0.05 vs.BCG)。受单个蛋白Ag85B和ESAT6刺激以后,融合蛋白ESAT6-Ag85B组产生的IFN-y量与对照BCG组相比,都有明显的差异(*p0.05vs. BCG)。最后,免疫后小鼠脾脏淋巴细胞受单个蛋白Ag85B和TB10.4特异性抗原表位刺激时,融合蛋白TB10.4-Ag85B组,产生的IFN-y量与对照BCG组相比,有明显的差异(*p0.05vs. BCG)。结论：成功构建了不带有标签的结核分枝杆菌融合蛋白ESAT6-Ag85B、TB10.4-Ag85B、ESAT6-TB8.4和TB10.4-TB8.4,且利用不同的色谱柱使融合蛋白得到了有效的纯化；动物免疫实验显示融合蛋白较单个抗原有更强的免疫原性。上述融合蛋白将用于构建结核亚单位候选疫苗,进一步评价其免疫保护效果。
[Abstract]:1 . Study on the correlation between the pathological model of skin liquefaction and the virulence of Mycobacterium tuberculosis in rabbits

Objective : To investigate the effects of BCG , Mycobacterium smegmatis ( M.smegmatis ) and H37Ra complementary strain ( H37Ra strain , respectively ) of BCG , Mycobacterium smegmatis ( M.smegmatis ) and H37Ra complementary strain ( H37Ra strain of 11 different sources of H37Rv wild - type gene ) on the skin of rabbits .

Methods : The rabbits were randomly divided into 6 groups : BCG , H37Ra and M . smegmatis were injected intracutaneously with 5 脳 106 CFU BCG , H37Ra and M . smegmatis .
In the experiment , rabbits were randomly divided into 13 groups , H37Ra strain and pOLYG vector group were randomly divided into 13 groups , H37Ra strain and pOLYG vector group were used as control group , and the experimental group was H37Ra complementary strain group including P10 , P11 , P12 . The rabbits were divided into three groups : high dose ( 5x107 CFU ) , medium dose ( 5 脳 106 CFU ) and low dose ( 5x105 CFU ) . After 6 weeks , the rabbits were immunized again to observe the skin injury of rabbits .

Results : After the rabbits were inoculated with BCG , H37Ra and M . smegmatis ' s live bacterial solution and inactivated bacterial solution , the rabbits were able to observe the obvious inflammatory reaction and abscess formation and liquefaction and ulcer of the rabbits .
There was no obvious pathological change in the skin of rabbits . After re - immunization , it was found that the degree of pathological response in rabbits could be increased . The severity of the lesions in rabbits caused by BCG was the strongest , followed by H37Ra and M . smegmatis . The results showed that H37Ra complementary strain P10 ( 5 脳 107 CFU ) and medium dose group ( 5 脳 106 CFU ) could induce the maximal pathological changes of skin . However , the response of P11 ( phoP ) , P12 ( marR family ) , P16 ( nadD ) : and P16 ( nadD ) : and P20 ( ilvD ) were similar to those of vector control pOLYG and H37Ra . Compared with other groups , P18 ( nrdH ) and pknH could induce the most intense liquefaction reaction in rabbit skin lesions ;
There was no significant difference between the liquefaction intensity of P10 ( pstAl ) , P11 ( phoP ) , p13 ( lusR / uhpA family ) , P16 ( nadD ) , and P20 ( ilvD ) group compared with control group pOLYG and H37Ra .

Conclusion : BCG , H37Ra and M . smegmatis of the dose ( 5 脳 106 CFU ) can cause liquefaction in the skin of rabbits . Among them , the response of BCG group is the strongest , followed by H37Ra group , the weakest is the M . smegulate group . When the mycobacterium is inactivated , the ability to initiate liquefaction reaction is lost .

2 . Cloning , construction and immunogenicity detection of non - labeled tuberculosis fusion protein ESAT6 - Ag85B / TB10.4 - Ag85B / ESAT6 - TB8.4 / TB10.4 - TB8.4

Objective : To clone the fusion protein ESAT6 - Ag85B / TB10.4 - Ag85B / ESAT6 - TB8.4 / TB10.4 - TB8.4 and evaluate the immunogenicity of the fusion protein by animal experiments .

Methods : The recombinant plasmid pET30a - ESAT6 - Ag85B / pET30a - ESAT6 - TB8.4 / pET30a - TB10.4 - TB8.4 was designed by using molecular cloning technique . The recombinant plasmid pET30a - ESAT6 - Ag85B / pET30a - ESAT6 - TB8.4 / pET30a - TB10.4 - TB8.4 was prepared by using H37Rv - DNA as template .

Results : The ESAT6 , TB10.4 , TB8.4 and Ag85B sequences obtained by PCR amplification were completely consistent with GenBank . The expression products of fusion protein in E . coli were in agreement with the expected size , the molecular weight of fusion protein ESAT6 - Ag85B was about 38KD , the molecular weight of fusion protein TB10.4 - Ag85B was about 42KD , both of which were expressed in inclusion bodies .
The fusion protein ESAT6 - TB8.4 has a molecular weight of about 18KD , and the fusion protein TB10.4 - TB8.4 has a molecular weight of about 19KD . The fusion protein TB10.4 - TB8.4 has a molecular weight of about 19KD , and the two soluble proteins can be purified by using ion exchange chromatography and hydrophobic chromatography . The ELISA detection results show that the IFN - y produced by the fusion protein ESAT6 - TB8.4 group is higher than that of BCG and ESAT6 ( ** p0.05 vs . BCG and ESAT6 ) . Compared with BCG and TB8.4 groups , the levels of IFN - y were higher in TB10.4 - TB8.4 group than in BCG group and TB8.4 group ( ** p . 05 vs . BCG and TB8.4 ) , while IFN - 纬 levels were higher in TB10.4 - TB8.4 group than in BCG group ( p . 05 vs . BCG ) . After stimulation with single protein Ag85B and ESAT6 , the amount of IFN - y produced by the fusion protein ESAT6 - Ag85B group was significantly different from that of the control BCG group ( * p0.05 vs . BCG ) . Finally , when the spleen lymphocytes of the immunized mice were stimulated by single protein Ag85B and TB10.4 specific antigen epitopes , the fusion protein TB10.4 - Ag85B group showed a significant difference compared with the control BCG group ( * p0.05 vs . BCG ) .

Conclusion : The fusion protein ESAT6 - Ag85B , TB10.4 - Ag85B , ESAT6 - TB8.4 and TB10.4 - TB8.4 were successfully constructed .
The fusion protein will be used to construct the tuberculosis subunit vaccine and further evaluate its immune protection effect .

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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