發(fā)布時(shí)間：2018-04-19 01:14

  本文選題：致倦庫(kù)蚊 + 天蠶素基因家族　； 參考：《貴州醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:克隆致倦庫(kù)蚊Cecropin基因家族的4個(gè)基因;比較分析Cecropin家族基因的結(jié)構(gòu)、氨基酸序列、蛋白理化特性,預(yù)測(cè)蛋白空間結(jié)構(gòu),以及其進(jìn)化關(guān)系;了解Cecropin家族基因在致倦庫(kù)蚊不同發(fā)育時(shí)期的表達(dá)譜;選擇Cec B2進(jìn)行原核表達(dá),檢測(cè)Cec B2多肽的抑菌活性。方法:通過生物信息學(xué)方法從致倦庫(kù)蚊基因組和轉(zhuǎn)錄組中篩選出致倦庫(kù)蚊Cecropin基因家族的4個(gè)基因,并進(jìn)行序列分析和系統(tǒng)發(fā)育分析;采用RT-PCR方法從致倦庫(kù)蚊c DNA中擴(kuò)增出Cecropin家族4個(gè)基因,與p MD-18T載體連接后測(cè)序鑒定;提取致倦庫(kù)蚊不同發(fā)育時(shí)期的總RNA,通過半定量PCR檢測(cè)4個(gè)Cecropin基因在不同齡期的表達(dá);構(gòu)建Cec B2重組表達(dá)質(zhì)粒,鑒定正確后轉(zhuǎn)入Rosetta表達(dá)菌中,優(yōu)化IPTG誘導(dǎo)濃度和誘導(dǎo)時(shí)間,表達(dá)純化重組蛋白,并采用SDS-PAGE和Western blot對(duì)重組蛋白進(jìn)行檢測(cè)和鑒定,進(jìn)一步透析、濃縮及腸激酶切割純化的融合蛋白,采用Tricine-SDS-PAGE檢測(cè)Cec B2目的蛋白。同時(shí)通過化學(xué)合成方法合成足量Cec B2成熟肽,并進(jìn)行體外抑菌活性檢測(cè)。結(jié)果:1.致倦庫(kù)蚊Cecropin基因家族的編碼氨基酸平均長(zhǎng)度約60aa,編碼蛋白的平均理論分子量和平均等電點(diǎn)p I值分別為6389.0和10.69,為小分子堿性蛋白;系統(tǒng)進(jìn)化分析表明致倦庫(kù)蚊Cecropin與岡比亞按蚊和埃及伊蚊親緣關(guān)系較近。2.半定量PCR研究發(fā)現(xiàn),Cec B2在整個(gè)發(fā)育時(shí)期均有表達(dá),而Cec B1,Cec A1,Cec A2除卵期無(wú)明顯表達(dá)外其余時(shí)期均有不同程度表達(dá),總的表達(dá)趨勢(shì)為隨蟲齡的增長(zhǎng)而增加。3.成功構(gòu)建原核表達(dá)載體p ET32a-Cec B2,最佳表達(dá)條件為:0.05mmol/L IPTG誘導(dǎo)3h,表達(dá)純化獲得大小約25k D的融合蛋白,Western blot鑒定融合蛋白可與鼠抗His-tag單克隆抗體發(fā)生抗原抗體結(jié)合反應(yīng),證明成功獲得Cec B2融合蛋白,但本研究獲得融合蛋白不具有抑菌活性。4.化學(xué)合成Cec B2多肽分別對(duì)G+菌(金黃色葡萄球菌、白色葡萄球菌、溶壁微球菌),G-菌(大腸埃希菌)以及真菌(白色念珠菌)均表現(xiàn)出不同程度的抑菌活性,細(xì)菌最大抑菌圈達(dá)到17mm(溶壁微球菌),真菌抑菌圈達(dá)到8mm(白色念珠菌)。結(jié)論:1.致倦庫(kù)蚊Cecropin為小分子堿性蛋白。2.Cec B2在整個(gè)發(fā)育時(shí)期均有表達(dá),Cec B1,Cec A1,Cec A2除卵期無(wú)明顯表達(dá)外其余時(shí)期均有不同程度表達(dá)。3.成功構(gòu)建p ET32a-Cec B2原核表達(dá)載體,獲取了大小約25k D的可溶性融合蛋白。4.Cec B2合成多肽對(duì)G+菌,G-菌以及真菌(白色念珠菌)具有不同程度抑菌作用,推測(cè)其具有潛在的抗生素研發(fā)意義。
[Abstract]:Objective: to clone four genes of Cecropin gene family of Culex pipiens quinquefasciatus, compare and analyze the structure, amino acid sequence, physicochemical properties of protein, predict the spatial structure of protein and its evolutionary relationship.To investigate the expression profiles of Cecropin family genes in different developmental stages of Culex pipiens quinquefasciatus, Cec B2 was selected for prokaryotic expression and the bacteriostatic activity of Cec B2 polypeptide was detected.Methods: four genes of Cecropin gene family of Culex pipiens quinquefasciatus were screened by bioinformatics from the genome and transcriptome of Culex pipiens quinquefasciatus, and sequenced and phylogenetic analysis were performed.Four Cecropin family genes were amplified from c DNA of Culex pipiens quinquefasciatus by RT-PCR method and sequenced with p MD-18T vector. The total RNAs of Culex quinquefasciatus at different developmental stages were extracted, and the expression of four Cecropin genes at different ages were detected by semi-quantitative PCR.The recombinant expression plasmid of Cec B2 was constructed, identified correctly and then transferred into Rosetta expression strain. The recombinant protein was expressed and purified by optimizing the concentration and time of IPTG induction. The recombinant protein was detected and identified by SDS-PAGE and Western blot for further dialysis.The fusion protein was purified by concentration and enterokinase cleavage. Cec B2 target protein was detected by Tricine-SDS-PAGE.At the same time, the mature peptide of Cec B2 was synthesized by chemical synthesis method, and the antibacterial activity was tested in vitro.The result is 1: 1.The average length of encoded amino acids in the Cecropin gene family of Culex pipiens quinquefasciatus was about 60aa, and the average theoretical molecular weight and average isoelectric point (Pi) of the encoded proteins were 6389.0 and 10.69, respectively.Phylogenetic analysis showed that Cecropin was closely related to Anopheles gambiae and Aedes aegypti.The results of semi-quantitative PCR showed that Cec B2 was expressed during the whole development period, while Cec B1Cec A1CA2 was expressed in different degrees at all stages except in egg stage, and the total expression trend was increased with the increase of worm age.The prokaryotic expression vector p ET32a-Cec B2 was successfully constructed. The optimal expression conditions were as follows: 0. 05 mmol / L IPTG was induced for 3 h. The fusion protein was purified and purified by Western blot. The fusion protein was identified to react with mouse anti His-tag monoclonal antibody.It was proved that the fusion protein of Cec B2 was successfully obtained, but the fusion protein had no bacteriostatic activity. 4.The chemically synthesized Cec B2 peptides showed different degrees of bacteriostatic activity against G bacteria (Staphylococcus aureus, Staphylococcus albicans, Micrococcus micrococci G- (Escherichia coli) and fungi (Candida albicans), respectively.The maximum bacteriostasis of bacteria was 17mm (micrococcus lysoid, 8mm) (Candida albicans).Conclusion 1.The Cecropin of Culex pipiens quinquefasciatus was a small molecular basic protein. 2. Cec B2 was expressed in different degrees during the whole development period except for the egg stage.The prokaryotic expression vector of p ET32a-Cec B2 was successfully constructed, and the soluble fusion protein of about 25kD was obtained. 4. The synthetic peptide of Cec B2 had different degree of bacteriostatic effect on G- and fungi (Candida albicans).It is speculated that it has potential significance in antibiotic research and development.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78

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