本文選題：結(jié)核分枝桿菌 + 病理　； 參考：《蘭州大學》2011年碩士論文

【摘要】：1.家兔皮膚液化病理模型與分枝桿菌毒力差異相關(guān)性研究 目的：利用卡介苗BCG、結(jié)核分枝桿菌減毒株H37Ra、恥垢分枝桿菌Mycobacterium smegmatis(M. smegmatis)以及H37Ra互補株(分別重組了11種來源H37Rv野生型基因的H37Ra菌株)感染家兔皮膚,觀察皮膚病理變化情況,可以快速有效地評價不同分枝桿菌毒力以及引起家兔皮膚液化能力的差異。 方法：實驗一中家兔隨機分成6組,分別進行腹部兩側(cè)皮內(nèi)注射5×106CFU的BCG、H37Ra和M. smegmatis的活菌液和熱滅活菌液；距初次免疫后約42天,每組以相同劑量的同種菌液對家兔進行再次皮內(nèi)免疫,觀察家兔皮膚病理變化。實驗二中家兔隨機分成13組,設(shè)立H37Ra株和pOLYG載體組為對照組,實驗組為H37Ra互補株組包括P10、P11、P12…P20組。以上菌株分別感染家兔皮膚,每組注射劑量分為三種即高劑量(5x107CFU)、中劑量(5×106CFU)、低劑量(5x105CFU)。六周后進行再次免疫,觀察家兔皮膚損傷情況。 結(jié)果：實驗一,家兔分別經(jīng)皮內(nèi)接種BCG、H37Ra和M. smegmatis的活菌液和熱滅活菌液后,活菌組家兔可以觀察到皮膚有明顯的炎癥反應和膿腫形成以及液化、潰瘍的發(fā)生；而熱滅活組都不能引起家兔皮膚發(fā)生明顯的病理變化。再次免疫后可以發(fā)現(xiàn)各組病理反應程度會加重�；罹M引起家兔皮膚病變的嚴重程度為：BCG引起的病變反應最強,其次是H37Ra, M. smegmatis的反應最弱。實驗二,H37Ra互補株感染家兔皮膚以后,高劑量組(5x107CFU)和中劑量組(5×106CFU)均可誘導產(chǎn)生強烈的家兔皮膚病理改變。對于不同H37Ra互補株,所引起的病灶反應程度存在差異：H37Ra互補株P(guān)10(重組了H37Rv的基因pstAl)引起的病灶體積最大,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)與載體對照pOLYG和H37Ra引起的病灶反應比較相似。與其他組相比,P18(nrdH)和P14(pknH)能促使家兔皮膚病灶發(fā)生最強烈的液化反應；P10(pstAl)、P11(phoP)、P13(luxR/uhpA family)、P16(nadD)和P20(ilvD)組的液化強度與對照組pOLYG和H37Ra比較沒有明顯差異。 結(jié)論：劑量(5×106 CFU)的活菌BCG、H37Ra和M.smegmatis能夠引起家兔皮膚發(fā)生液化現(xiàn)象,其中BCG組病灶反應最強烈,其次是H37Ra組,最弱為M.smegmatis組。當分枝桿菌被滅活后,便喪失了引發(fā)液化反應的能力。而且利用這種家兔皮膚模型可以有效評價不同H37Ra互補株引起的病理變化的差異。 2.無標簽結(jié)核融合蛋白ESAT6-Ag85B/TB10.4-Ag85B/ESAT6-TB8.4/ TB10.4-TB8.4的克隆構(gòu)建和免疫原性檢測 目的：克隆構(gòu)建無標簽結(jié)核分枝桿菌融合蛋白ESAT6-Ag85B/TB10.4-Ag85B/ESAT6-TB8.4/TB10.4-TB8.4,并利用動物實驗對此融合蛋白的免疫原性進行初步評價。 方法：應用分子克隆技術(shù)設(shè)計含有不同酶切位點的ESAT6、TB10.4、TB8.4和Ag85B的引物,以H37Rv-DNA為模板PCR擴增出相應大小的基因片段,通過基因工程技術(shù)進行重組而獲得重組質(zhì)粒pET30a-ESAT6-Ag85B/pET30a-TB10.4-Ag85B/pET30a-ESAT6-TB8.4/pET30a-TB10.4-TB8.4。將重組質(zhì)粒轉(zhuǎn)化入大腸桿菌菌株BL21,進行IPTG誘導表達,采用IEX(離子交換色譜層析)和HIC(疏水作用色譜層析)兩種方法進行融合蛋白的純化。最后將蛋白與佐劑DDA混合配制成蛋白疫苗進行動物實驗,將C57BL/6小鼠隨機分成9組,設(shè)立PBS、BCG和單個抗原作為對照組,融合蛋白組為實驗組。免疫后第14周采用ELISA技術(shù)檢測免疫后小鼠的脾臟淋巴細胞分泌INF-y水平。 結(jié)果：PCR擴增獲得的ESAT6、TB10.4、TB8.4和Ag85B序列與GenBank中完全一致。融合蛋白在大腸桿菌中表達產(chǎn)物與預計大小相吻合,融合蛋白ESAT6-Ag85B分子量約38KD、融合蛋白TB10.4-Ag85B分子量約42KD,二者以包涵體的形式表達,最后應用離子交換層析和疏水層析可純化此兩種復性后的蛋白；融合蛋白ESAT6-TB8.4分子量約18KD、融合蛋白TB10.4-TB8.4分子量約19KD,二者以上清液表達為主,應用離子交換層析和疏水層析可純化此兩種可溶性蛋白。ELISA檢測結(jié)果顯示：免疫后小鼠脾臟淋巴細胞受單個蛋白ESAT6和TB8.4刺激以后,融合蛋白ESAT6-TB8.4組產(chǎn)生的IFN-y高于BCG組和ESAT6組(**p0.05 vs.BCG and ESAT6)。與BCG組、TB8.4組相比,當受單個蛋白TB8.4刺激以后,融合蛋白TB10.4-TB8.4組分泌IFN-y水平高于BCG組和TB8.4組(**p0.05 vs.BCG and TB8.4);而針對TB10.4特異性抗原表位刺激時,融合蛋白TB10.4-TB8.4組分泌IFN-γ水平高于BCG組(p0.05 vs.BCG)。受單個蛋白Ag85B和ESAT6刺激以后,融合蛋白ESAT6-Ag85B組產(chǎn)生的IFN-y量與對照BCG組相比,都有明顯的差異(*p0.05vs. BCG)。最后,免疫后小鼠脾臟淋巴細胞受單個蛋白Ag85B和TB10.4特異性抗原表位刺激時,融合蛋白TB10.4-Ag85B組,產(chǎn)生的IFN-y量與對照BCG組相比,有明顯的差異(*p0.05vs. BCG)。 結(jié)論：成功構(gòu)建了不帶有標簽的結(jié)核分枝桿菌融合蛋白ESAT6-Ag85B、TB10.4-Ag85B、ESAT6-TB8.4和TB10.4-TB8.4,且利用不同的色譜柱使融合蛋白得到了有效的純化；動物免疫實驗顯示融合蛋白較單個抗原有更強的免疫原性。上述融合蛋白將用于構(gòu)建結(jié)核亞單位候選疫苗,進一步評價其免疫保護效果。
[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 - 緯 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 .

【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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