本文關鍵詞： 結(jié)核分枝桿菌 Esat6 人集落刺激因子 融合質(zhì)粒 DNA疫苗　出處：《暨南大學》2007年碩士論文　論文類型：學位論文

【摘要】： 目的： 從質(zhì)粒pVAE中克隆目的基因Esat6，與hGM-CSF一起構建雙順反子表達載體pIEG，進而轉(zhuǎn)染Hela細胞，并進行鑒定。為進一步研究具有預防性和治療性抗結(jié)核桿菌新型核酸疫苗奠定了基礎。 方法： 1．應用分子克隆技術，，以質(zhì)粒pVAE為模板PCR擴增出Esat6基因，與pIRES載體的MCS A進行重組，構建pIEsat6重組質(zhì)粒。再以pORF-hGM-CSF為模板PCR擴增出hGM-CSF基因，與pIRES載體的MCS B進行重組，構建pIGM雙順反子重組質(zhì)粒；2．上述兩重組質(zhì)粒經(jīng)多種分子生物學方法進行鑒定后，再通過酶切、連接，將hGM-CSF構建于pIEsat6質(zhì)粒中，形成雙順反子融合表達載體pIEG；3．將pIEG轉(zhuǎn)染人宮頸癌細胞(Hela)；4．通過SDS-PAGE蛋白電泳鑒定細胞內(nèi)Esat6基因的表達情況，并拍照；5．通過免疫組織化學方法檢測細胞內(nèi)Esat6基因的表達情況，并拍照；6．通過ELISA方法檢測細胞中hGM-CSF基因的表達情況。 結(jié)果： 1．通過PCR可克隆得到相應大小的產(chǎn)物；酶切鑒定所切下的兩片段大小約為0.29 kb以及0.47 kb，與預計相符；測序結(jié)果與報道一致；2．SDS-PAGE電泳能檢測到細胞內(nèi)有Esat6蛋白的表達；3．免疫組織化學方法能檢測到表達Esat6的陽性細胞；4．ELISA方法能檢測到細胞培養(yǎng)上清中hGM-CSF的量與陰性對照組存在顯著性差異。 結(jié)論： 成功克隆并構建了含有結(jié)核分枝桿菌Esat6基因與hGM-CSF基因的雙順反子融合表達載體，并在Hela細胞中成功表達。為研制優(yōu)于BCG的抗結(jié)核病的防治療性DNA疫苗奠定了基礎。
[Abstract]:Objective: The target gene Esat6 was cloned from the plasmid pVAE, and the double cistronic expression vector pIEG was constructed together with hGM-CSF, and then transfected into Hela cells. The results provide a basis for the further study of novel nucleic acid vaccine with preventive and therapeutic anti-tuberculosis. Methods: 1. Using molecular cloning technique, the Esat6 gene was amplified by PCR using plasmid pVAE as template, and was recombined with MCS A of pIRES vector. The pIEsat6 recombinant plasmid was constructed and the hGM-CSF gene was amplified by PCR using pORF-hGM-CSF as the template. The hGM-CSF gene was recombined with MCS B of pIRES vector. Construction of pIGM double cistron recombinant plasmid; 2.After the two recombinant plasmids were identified by many molecular biological methods, hGM-CSF was constructed into pIEsat6 plasmid by enzyme digestion and ligation. A double cistron fusion expression vector pIEG was formed. 3. Transfection of pIEG into human cervical carcinoma cell line HelaHN; 4. SDS-PAGE protein electrophoresis was used to identify the expression of Esat6 gene in the cells. 5. The expression of Esat6 gene in the cells was detected by immunohistochemical method, and the pictures were taken. 6. The expression of hGM-CSF gene was detected by ELISA. Results: 1. The products of corresponding size can be obtained by PCR cloning. The size of the two fragments was about 0.29 kb and 0.47 kb, which was consistent with the expected size. The results of sequencing were consistent with the report. 2. The expression of Esat6 protein was detected by SDS-PAGE. 3. The positive cells expressing Esat6 could be detected by immunohistochemistry. 4. The amount of hGM-CSF in the supernatant of cell culture was significantly different from that in the negative control group by Elisa. Conclusion: The expression vector containing Esat6 gene and hGM-CSF gene of Mycobacterium tuberculosis was cloned and constructed successfully. It was successfully expressed in Hela cells, which laid a foundation for the development of a therapeutic DNA vaccine against tuberculosis, which was superior to BCG.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R392

【參考文獻】

相關期刊論文 前10條

1 王慶敏,胡振林,周鳳娟,肖存杰,章建程,孫樹漢;ESAT6抗原DNA疫苗在小鼠體內(nèi)誘導的免疫應答[J];第二軍醫(yī)大學學報;2004年06期

2 師長宏,范雄林,柏銀蘭,薛瑩,張海,徐志凱;結(jié)核分枝桿菌Ag85B-ESAT6融合蛋白在小鼠體內(nèi)誘導的免疫應答及其保護力[J];第四軍醫(yī)大學學報;2004年18期

3 陳峻崧,竇駿,趙楓姝,陳國兵,房雪峰,洪曉武,唐權;結(jié)核桿菌Ag85A和mGM-CSF共同表達載體的構建與CTL活性的誘導[J];現(xiàn)代免疫學;2004年05期

4 顧田園,蔡宏,田霞,余大海,朱玉賢;結(jié)核分枝桿菌四價DNA疫苗免疫原性和保護效率研究[J];生物化學與生物物理進展;2005年04期

5 史小玲,李暉,鐘森,張建軍,鄧存良,黃永茂,盧潤生,吳建林,衛(wèi)幫富,蔣紹雙;Hsp70/CD80嵌合DNA疫苗對結(jié)核桿菌的治療作用[J];中華傳染病雜志;2004年01期

6 江山,朱道銀,駱旭東,蔣英,陳全;結(jié)核分枝桿菌DNA疫苗對小鼠結(jié)核病免疫治療作用的實驗研究[J];中華結(jié)核和呼吸雜志;2005年05期

7 賈克明,鄧濤;DNA疫苗[J];中華內(nèi)科雜志;1998年07期

8 關菁,馬俐君,魏麗惠;HSV-TK與GM-CSF基因雙順反子逆轉(zhuǎn)錄病毒載體轉(zhuǎn)染卵巢癌細胞體外特性的研究(英文)[J];Chinese Medical Journal;2001年02期

9 駱旭東,朱道銀,江山,陳全,蔣英;Ag85B-MPT64融合基因疫苗對鼠結(jié)核分枝桿菌感染的保護作用[J];中華醫(yī)學雜志;2004年08期

10 李暉,李榕,鐘森,任紅;結(jié)核桿菌Mtb8.4/hIL12嵌合基因真核表達質(zhì)粒的構建與表達[J];中國人獸共患病雜志;2005年03期

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