胸膜肺炎放線桿菌Lip40蛋白生物學(xué)特性研究
發(fā)布時(shí)間：2018-08-24 16:34
【摘要】：胸膜肺炎放線桿菌(A ctinobacillus pleuropneumoniae, APP)是引起豬胸膜肺炎(Porcine pleuropneumonia)的病原菌,該病是一種嚴(yán)重的豬呼吸道疾病且具有致死性,給養(yǎng)豬業(yè)造成了極大的經(jīng)濟(jì)損失。到目前為止,已經(jīng)發(fā)現(xiàn)了15種APP血清型。APP的感染以及致病過程中有許多毒力因子的參與,包括RTX毒素、莢膜多糖、脂多糖、粘附因子、蛋白酶、外膜蛋白以及轉(zhuǎn)錄調(diào)控因子等。本實(shí)驗(yàn)通過多種生物信息學(xué)分析軟件,預(yù)測(cè)發(fā)現(xiàn)APP中共有60種脂蛋白,并從中挑選出一個(gè)具有特殊串聯(lián)重復(fù)序列Q(E/D/P)QPK的脂蛋白Lip40。統(tǒng)計(jì)發(fā)現(xiàn)不同血清型APP菌株中,該重復(fù)序列的重復(fù)次數(shù)不同。通過BioEdit (7.0 版)對(duì)Lip40蛋白進(jìn)行多序列比對(duì)分析發(fā)現(xiàn),其與多種已知的脂蛋白和Tbp蛋白具有較高的相似性|。利用SWISS-model對(duì)其三維結(jié)構(gòu)進(jìn)行預(yù)測(cè)分析,結(jié)果顯示Lip40蛋白由1個(gè)p筒(該β筒由8條p折疊構(gòu)成)以及4條p折疊所構(gòu)成,并通過WinCoot軟件將Lip40蛋白分別與APP TbpB N lobe、C lobe進(jìn)行三維結(jié)構(gòu)重疊比對(duì),發(fā)現(xiàn)Lip40蛋白與兩者的三維結(jié)構(gòu)都具有較高的相似性。為了獲得重組Lip40蛋白,本研究首先通過PCR克隆到APP SLW01菌株中的lip40基因,目的基因經(jīng)限制性內(nèi)切酶酶切后,連入pGEX-KG載體,轉(zhuǎn)化E.coli BL21感受態(tài)細(xì)胞,經(jīng)IPTG誘導(dǎo)獲得重組蛋白rLip40。經(jīng)免疫印跡分析,rLip40泳道上出現(xiàn)一個(gè)分子量約為56 kDa的雜交帶,表明抗APP的陽性血清可以特異性的識(shí)別并結(jié)合rLip40蛋白,說明rLip40蛋白具有較好的免疫反應(yīng)性。另外,本研究以rLip40蛋白為免疫原免疫大白兔,獲得兔抗rLip40多克隆抗體,其效價(jià)可達(dá)1：2×105,表明rLip40蛋白可以誘導(dǎo)動(dòng)物機(jī)體產(chǎn)生較強(qiáng)的免疫反應(yīng),說明Lip40蛋白具有良好的免疫原性。為鑒定Lip40蛋白的亞細(xì)胞定位,本研究分別提取APP各亞細(xì)胞蛋白組分,以兔抗rLip40蛋白多克隆抗體為一級(jí)抗體,通過免疫印跡方法對(duì)Lip40蛋白進(jìn)行亞細(xì)胞定位,結(jié)果發(fā)現(xiàn)其定位于外膜,與之前生物信息學(xué)結(jié)果相同。之后本研究分析了Lip40蛋白在不同應(yīng)激條件下的表達(dá)情況,結(jié)果顯示在高溫、厭氧和低溫等應(yīng)激條件下,Lip40蛋白在轉(zhuǎn)錄水平上和翻譯水平上均呈現(xiàn)上調(diào),故認(rèn)為L(zhǎng)ip40蛋白是一個(gè)多重應(yīng)激響應(yīng)因子,并推測(cè)Lip40蛋白可能是APP的毒力因子,可能參與病原菌-環(huán)境以及病原菌-宿主之間的相互作用。本研究分析了Lip40蛋白的免疫保護(hù)性,以rLip40蛋白為免疫原,免疫小鼠,隨后以致死劑量的高致病性APP菌株攻毒,結(jié)果表明Lip40蛋白對(duì)小鼠的保護(hù)率達(dá)到75%,雖然其保護(hù)率低于ApxIA毒素蛋白(100%)和滅活疫苗(91.7%),但顯著高于空白對(duì)照組。因此,Lip40蛋白可以作為高效APP疫苗研發(fā)的候選蛋白。本研究還以前期構(gòu)建的lip40基因缺失突變株△lip40為親本,成功構(gòu)建了回復(fù)突變株CΔlip40,并對(duì)其生物學(xué)特性進(jìn)行了初步分析。穿梭質(zhì)粒pJFF-lip40可在APP中穩(wěn)定遺傳,并且未對(duì)APP生長(zhǎng)能力造成顯著影響。此外,APP SLW01菌株和△lip40對(duì)氯霉素高度敏感,但回復(fù)突變株CAlip40較前兩種菌株對(duì)氯霉素抗性明顯增強(qiáng),表明穿梭質(zhì)粒pJFF224-XN抗性基因可在APP菌株中穩(wěn)定表達(dá)�；貜�(fù)突變株CΔlip40的成功構(gòu)建為分析Lip40蛋白的致病機(jī)制提供了有效材料,本實(shí)驗(yàn)中建立的APP互補(bǔ)菌株構(gòu)建系統(tǒng)以及APP抗性評(píng)價(jià)方法將有助于今后研究APP生物學(xué)特性及基因功能。
[Abstract]:A ctinobacillus pleuropneumoniae (APP) is a pathogen causing porcine pleuropneumonia (Porcine pleuropneumonia). This disease is a serious * * * respiratory disease and is lethal. It has caused great economic losses to the pig industry. Up to now, 15 APP serotype.APP infections have been detected and caused. There are many virulence factors involved in the pathogenesis, including RTX toxin, capsular polysaccharide, lipopolysaccharide, adhesion factor, protease, outer membrane protein and transcription regulator. Through a variety of bioinformatics analysis software, we predicted that there were 60 kinds of lipoproteins in APP, and selected a special tandem repeat Q (E/D/P) QPK. Lip40. It was found that the repeats were different in different serotypes of APP strains. BioEdit (version 7.0) analysis showed that Lip40 had high similarity with many known lipoproteins and Tbp proteins. The results showed that Lip40 protein was composed of one p-tube (the beta-tube was composed of eight p-folds) and four p-folds. The three-dimensional structure overlap of Lip40 protein with APP TbpB N lobe and CLOBE was compared by WinCoot software. It was found that the three-dimensional structure of Lip40 protein was similar to that of APP TbpB N lobe and CLOB. The lip40 gene of APP SLW01 strain was cloned by PCR. After restriction endonuclease digestion, the target gene was inserted into pGEX-KG vector and transformed into E.coli BL21 competent cells. The recombinant protein rLip40 was obtained by IPTG induction. A hybridization band with a molecular weight of about 56 kDa appeared on the swimming track of rLip40, indicating that the positive serum against APP could be obtained. In addition, the polyclonal antibody against rLip40 was obtained by immunizing rabbits with rLip40 protein. The titer of the polyclonal antibody was 1:2 X 105, indicating that rLip40 protein could induce strong immune response in vivo, indicating that the Lip40 protein possessed the characteristics of immunoreactivity. In order to identify the subcellular localization of Lip40 protein, the subcellular localization of Lip40 protein was performed by immunoblotting with rabbit anti-rLip40 polyclonal antibody as the primary antibody. The results showed that the subcellular localization of Lip40 protein was in the adventitia, which was the same as the previous bioinformatics results. This study analyzed the expression of Lip40 protein under different stress conditions. The results showed that Lip40 protein was up-regulated at transcriptional and translation levels under high temperature, anaerobic and hypothermic stress conditions. Therefore, Lip40 protein was considered to be a multiple stress response factor, and Lip40 protein might be a virulent factor of APP, possibly a reference. Interaction with pathogen-environment and pathogen-host. This study analyzed the immunoprotective effect of Lip40 protein. Mice were immunized with rLip40 protein as an immunogen, and then infected with high pathogenic APP strain at lethal doses. The results showed that the protective rate of Lip40 protein in mice was 75%, although the protective rate was lower than that of ApxIA toxin protein. Lip40 protein can be used as a candidate protein for the development of high-efficiency APP vaccine. In this study, a reply mutant Clip40 was successfully constructed and its biological characteristics were preliminarily analyzed. JFF-lip40 could be inherited steadily in APP and had no significant effect on the growth ability of APP. In addition, strain APP SLW01 and delta-lip40 were highly sensitive to chloramphenicol, but the resistance of restored mutant CAlip40 to chloramphenicol was significantly stronger than that of the first two strains, suggesting that shuttle plasmid pJFF224-XN resistance gene could be stably expressed in APP strain. The successful construction of lip40 provides an effective material for analyzing the pathogenic mechanism of Lip40 protein. The construction system of complementary strains of APP and the evaluation method of APP resistance established in this study will be helpful to study the biological characteristics and gene function of APP in the future.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61
本文編號(hào)：2201388
[Abstract]:A ctinobacillus pleuropneumoniae (APP) is a pathogen causing porcine pleuropneumonia (Porcine pleuropneumonia). This disease is a serious * * * respiratory disease and is lethal. It has caused great economic losses to the pig industry. Up to now, 15 APP serotype.APP infections have been detected and caused. There are many virulence factors involved in the pathogenesis, including RTX toxin, capsular polysaccharide, lipopolysaccharide, adhesion factor, protease, outer membrane protein and transcription regulator. Through a variety of bioinformatics analysis software, we predicted that there were 60 kinds of lipoproteins in APP, and selected a special tandem repeat Q (E/D/P) QPK. Lip40. It was found that the repeats were different in different serotypes of APP strains. BioEdit (version 7.0) analysis showed that Lip40 had high similarity with many known lipoproteins and Tbp proteins. The results showed that Lip40 protein was composed of one p-tube (the beta-tube was composed of eight p-folds) and four p-folds. The three-dimensional structure overlap of Lip40 protein with APP TbpB N lobe and CLOBE was compared by WinCoot software. It was found that the three-dimensional structure of Lip40 protein was similar to that of APP TbpB N lobe and CLOB. The lip40 gene of APP SLW01 strain was cloned by PCR. After restriction endonuclease digestion, the target gene was inserted into pGEX-KG vector and transformed into E.coli BL21 competent cells. The recombinant protein rLip40 was obtained by IPTG induction. A hybridization band with a molecular weight of about 56 kDa appeared on the swimming track of rLip40, indicating that the positive serum against APP could be obtained. In addition, the polyclonal antibody against rLip40 was obtained by immunizing rabbits with rLip40 protein. The titer of the polyclonal antibody was 1:2 X 105, indicating that rLip40 protein could induce strong immune response in vivo, indicating that the Lip40 protein possessed the characteristics of immunoreactivity. In order to identify the subcellular localization of Lip40 protein, the subcellular localization of Lip40 protein was performed by immunoblotting with rabbit anti-rLip40 polyclonal antibody as the primary antibody. The results showed that the subcellular localization of Lip40 protein was in the adventitia, which was the same as the previous bioinformatics results. This study analyzed the expression of Lip40 protein under different stress conditions. The results showed that Lip40 protein was up-regulated at transcriptional and translation levels under high temperature, anaerobic and hypothermic stress conditions. Therefore, Lip40 protein was considered to be a multiple stress response factor, and Lip40 protein might be a virulent factor of APP, possibly a reference. Interaction with pathogen-environment and pathogen-host. This study analyzed the immunoprotective effect of Lip40 protein. Mice were immunized with rLip40 protein as an immunogen, and then infected with high pathogenic APP strain at lethal doses. The results showed that the protective rate of Lip40 protein in mice was 75%, although the protective rate was lower than that of ApxIA toxin protein. Lip40 protein can be used as a candidate protein for the development of high-efficiency APP vaccine. In this study, a reply mutant Clip40 was successfully constructed and its biological characteristics were preliminarily analyzed. JFF-lip40 could be inherited steadily in APP and had no significant effect on the growth ability of APP. In addition, strain APP SLW01 and delta-lip40 were highly sensitive to chloramphenicol, but the resistance of restored mutant CAlip40 to chloramphenicol was significantly stronger than that of the first two strains, suggesting that shuttle plasmid pJFF224-XN resistance gene could be stably expressed in APP strain. The successful construction of lip40 provides an effective material for analyzing the pathogenic mechanism of Lip40 protein. The construction system of complementary strains of APP and the evaluation method of APP resistance established in this study will be helpful to study the biological characteristics and gene function of APP in the future.
【學(xué)位授予單位】：華中師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.61
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