天堂国产午夜亚洲专区-少妇人妻综合久久蜜臀-国产成人户外露出视频在线-国产91传媒一区二区三区

幽門螺桿菌重組Bb-vacA-hpaA候選疫苗的構(gòu)建

發(fā)布時間:2018-07-24 21:08
【摘要】:目的 構(gòu)建幽門螺桿菌vacA-hpaA融合基因;構(gòu)建大腸桿菌-雙歧桿菌穿梭表達(dá)質(zhì)粒pGEX-vacA-hpaA;分析重組質(zhì)粒pGEX-vacA-hpaA在大腸桿菌BL21中的表達(dá);電穿孔轉(zhuǎn)化兩歧雙歧桿菌(Bb),構(gòu)建幽門螺桿菌重組Bb-vacA-hpaA候選疫苗。 方法 以質(zhì)粒pQE-vacA、pET-hpaA為模板,PCR擴(kuò)增獲得vacA和hpaA編碼基因序列;依次構(gòu)建重組質(zhì)粒pQE-hpaA, pQE-vacA-hpaA,以重組質(zhì)粒pQE-vacA-hpaA為模板,PCR擴(kuò)增構(gòu)建融合基因vacA-hpaA。將融合基因vacA-hpaA定向連接至大腸桿菌-雙歧桿菌穿梭表達(dá)載體pGEX-1λT,構(gòu)建重組質(zhì)粒pGEX-vacA-hpaA。電穿孔將pGEX-vacA-hpaA導(dǎo)入BL21, SDS-PAGE分析pGEX-vacA-hpaA在大腸桿菌BL21中的表達(dá);Western blot鑒定表達(dá)蛋白的抗原性。最后將pGEX-vacA-hpaA電轉(zhuǎn)化導(dǎo)入Bb,構(gòu)建幽門螺桿菌重組Bb-vacA-hpaA候選疫苗。 結(jié)果 瓊脂糖凝膠電泳證實(shí)vacA-hpaA融合基因全長1500bp左右,測序結(jié)果顯示其與預(yù)期結(jié)果一致。雙酶切證實(shí)vacA-hpaA融合基因成功插入pGEX-1λT質(zhì)粒,成功構(gòu)建重組質(zhì)粒pGEX-vacA-hpaA并成功轉(zhuǎn)入大腸桿菌BL21。SDS-PAGE結(jié)果顯示重組質(zhì)粒在大腸桿菌BL21中經(jīng)IPTG誘導(dǎo)4h表達(dá)了約85KDa的目的蛋白,Western blot分析顯示該蛋白能分別與兔抗VacA和兔抗HpaA免疫血清發(fā)生特異性結(jié)合。最后PCR及雙酶切鑒定證實(shí)重組質(zhì)粒pGEX-vacA-hpaA成功轉(zhuǎn)入兩歧雙歧桿菌Bb,成功獲得rBb-vacA-hpaA候選疫苗。 結(jié)論 1.成功構(gòu)建vacA-hpaA融合基因。 2.成功構(gòu)建幽門螺桿菌穿梭表達(dá)載體pGEX-vacA-hpaA。 3.幽門螺桿菌穿梭表達(dá)質(zhì)粒pGEX-vacA-hpaA能在大腸桿菌BL21中經(jīng)IPTG誘導(dǎo)表達(dá),而且所表達(dá)的重組蛋白同時具有VacA和HpaA蛋白單獨(dú)的抗原性。 4.成功構(gòu)建幽門螺桿菌rBb-vacA-hpaA候選疫苗。
[Abstract]:objective
To construct vacA-hpaA fusion gene of Helicobacter pylori, construct E. coli - Bifidobacterium shuttle expression plasmid pGEX-vacA-hpaA, analyze the expression of recombinant plasmid pGEX-vacA-hpaA in Escherichia coli BL21, electroporation transformation of Bifidobacterium Bifidobacterium (Bb), and construct a recombinant Bb-vacA-hpaA candidate vaccine for Helicobacter pylori.
Method
The plasmid pQE-vacA and pET-hpaA were used as templates to amplify the sequence of vacA and hpaA encoding gene, and the recombinant plasmid pQE-hpaA, pQE-vacA-hpaA, recombinant plasmid pQE-vacA-hpaA as the template, and PCR amplification and construction of the fusion gene vacA-hpaA. to connect the fusion gene vacA-hpaA directed to the Escherichia coli shuttle expression vector pGEX-1 lambda, were constructed by PCR amplification. To construct recombinant plasmid pGEX-vacA-hpaA. electroporation, pGEX-vacA-hpaA was introduced into BL21, SDS-PAGE was used to analyze the expression of pGEX-vacA-hpaA in Escherichia coli BL21; Western blot was used to identify the antigenicity of the expressed protein. Finally, pGEX-vacA-hpaA electrical conversion was transferred into Bb to construct a recombinant Bb-vacA-hpaA candidate vaccine for Helicobacter pylori.
Result
The agarose gel electrophoresis confirmed that the vacA-hpaA fusion gene was about 1500bp in length. The sequencing results showed that it was in agreement with the expected results. The double enzyme cut confirmed that the vacA-hpaA fusion gene was successfully inserted into the pGEX-1 lambda T plasmid, successfully constructed the recombinant plasmid pGEX-vacA-hpaA and successfully transferred to the Escherichia coli BL21.SDS-PAGE results to show that the recombinant plasmid was in Escherichia coli BL21. The target protein of about 85KDa was expressed by 4H by IPTG, and Western blot analysis showed that the protein could be specifically combined with Rabbit anti VacA and Rabbit anti HpaA immune sera. Finally, the identification of the recombinant plasmid pGEX-vacA-hpaA successfully transferred to Bifidobacterium Bifidobacterium Bb, and the rBb-vacA-hpaA candidate vaccine was successfully obtained by the identification of the Rabbit anti VacA and the Rabbit anti HpaA immune sera.
conclusion
1. the vacA-hpaA fusion gene was successfully constructed.
2. The shuttle expression vector pGEX-vacA-hpaA was successfully constructed.
3. Helicobacter pylori shuttle expression plasmid pGEX-vacA-hpaA can be induced by IPTG in Escherichia coli BL21, and the recombinant protein expressed at the same time has the antigenicity of VacA and HpaA protein alone.
4. Successful construction of candidate vaccine for Helicobacter pylori rBb-vacA-hpaA.
【學(xué)位授予單位】:大理學(xué)院
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2011
【分類號】:R392

【相似文獻(xiàn)】

相關(guān)會議論文 前10條

1 朱森林;陳e,

本文編號:2142658


資料下載
論文發(fā)表

本文鏈接:http://sikaile.net/xiyixuelunwen/2142658.html


Copyright(c)文論論文網(wǎng)All Rights Reserved | 網(wǎng)站地圖 |

版權(quán)申明:資料由用戶2d0ed***提供,本站僅收錄摘要或目錄,作者需要刪除請E-mail郵箱bigeng88@qq.com
亚洲男人的天堂就去爱| 熟女乱一区二区三区四区| 九九热国产这里只有精品| 麻豆欧美精品国产综合久久| 亚洲国产精品久久综合网| 中文字幕一区二区熟女| 一区二区三区日韩中文| 日韩高清中文字幕亚洲| 欧洲一区二区三区自拍天堂| 亚洲综合日韩精品欧美综合区| 亚洲一级在线免费观看| 偷自拍亚洲欧美一区二页| 国产人妻精品区一区二区三区| 日韩精品一区二区三区四区| 国产欧美日韩综合精品二区| 欧美久久一区二区精品| 欧美有码黄片免费在线视频| 亚洲中文字幕在线视频频道| 免费播放一区二区三区四区| 日韩综合国产欧美一区| 成人免费高清在线一区二区| 日韩熟妇人妻一区二区三区| 国产成人精品国产亚洲欧洲 | 亚洲综合香蕉在线视频| 欧美av人人妻av人人爽蜜桃 | 一区二区三区日韩中文| 五月激情婷婷丁香六月网| 妻子的新妈妈中文字幕| 中文字幕亚洲视频一区二区| 成年男女午夜久久久精品| 国产又猛又大又长又粗| 国产一级性生活录像片| 国产毛片对白精品看片| 欧美日韩亚洲巨色人妻| 久久99青青精品免费观看| 久久青青草原中文字幕| 熟女白浆精品一区二区| 黄色污污在线免费观看| 国产精品美女午夜福利| 中文字幕亚洲在线一区| 99久久国产精品免费|