中国人兽共患病杂志
中國人獸共患病雜誌
중국인수공환병잡지
CHINESE JOURNAL OF ZOONOSES
2004年
12期
1019-1023
,共5页
李振红%袁建平%居忠亮%赵蔚%李萍%胡宝瑜%时晓东%郭晓奎
李振紅%袁建平%居忠亮%趙蔚%李萍%鬍寶瑜%時曉東%郭曉奎
리진홍%원건평%거충량%조위%리평%호보유%시효동%곽효규
幽门螺杆菌%酵母双杂交%cDNA文库%LD-PCR
幽門螺桿菌%酵母雙雜交%cDNA文庫%LD-PCR
유문라간균%효모쌍잡교%cDNA문고%LD-PCR
Helicobacter pylori%yeast two-hybrid%cDNA library%LD-PCR
目的构建人胃黏膜细胞cDNA文库,并选取VacA毒素的p37片段将其克隆到pGBKT7以表达BD-p37融合蛋白作为诱饵.为进一步用所此诱饵来筛选所构建文库以其找到与VacA相互作用的蛋白奠定基础.方法用TRIzol试剂从胃腺癌病人手术切除的正常胃黏膜细胞中抽取总RNA经LD-PCR得到双链cDNA,参照Clontech 的MATCHMAKER Library Construction and Screening Kit 构建cDNA文库并用文库所含的独立克隆数和含插入片段的克隆数占总克隆数的比例来评价酵母双杂交文库的质量.用PCR从Helicobacter pylor基因组中扩增出p-37基因将其克隆入pGBKT7载体并使插入片段与GAL4 BD同框,插入片段的大小和序列的正确性由双酶切和测序得以证实.结果文库中共有1.9×106个独立克隆,含插入片段克隆所占比例为91.7%.双酶切和测序结果表明p37的正确插入,Western blot 结果证明了融合蛋白的表达.结论构建了较高质量的cDNA文库和与GAL4 BD融合的诱饵蛋白,为进一步的研究奠定了基础.
目的構建人胃黏膜細胞cDNA文庫,併選取VacA毒素的p37片段將其剋隆到pGBKT7以錶達BD-p37融閤蛋白作為誘餌.為進一步用所此誘餌來篩選所構建文庫以其找到與VacA相互作用的蛋白奠定基礎.方法用TRIzol試劑從胃腺癌病人手術切除的正常胃黏膜細胞中抽取總RNA經LD-PCR得到雙鏈cDNA,參照Clontech 的MATCHMAKER Library Construction and Screening Kit 構建cDNA文庫併用文庫所含的獨立剋隆數和含插入片段的剋隆數佔總剋隆數的比例來評價酵母雙雜交文庫的質量.用PCR從Helicobacter pylor基因組中擴增齣p-37基因將其剋隆入pGBKT7載體併使插入片段與GAL4 BD同框,插入片段的大小和序列的正確性由雙酶切和測序得以證實.結果文庫中共有1.9×106箇獨立剋隆,含插入片段剋隆所佔比例為91.7%.雙酶切和測序結果錶明p37的正確插入,Western blot 結果證明瞭融閤蛋白的錶達.結論構建瞭較高質量的cDNA文庫和與GAL4 BD融閤的誘餌蛋白,為進一步的研究奠定瞭基礎.
목적구건인위점막세포cDNA문고,병선취VacA독소적p37편단장기극륭도pGBKT7이표체BD-p37융합단백작위유이.위진일보용소차유이래사선소구건문고이기조도여VacA상호작용적단백전정기출.방법용TRIzol시제종위선암병인수술절제적정상위점막세포중추취총RNA경LD-PCR득도쌍련cDNA,삼조Clontech 적MATCHMAKER Library Construction and Screening Kit 구건cDNA문고병용문고소함적독립극륭수화함삽입편단적극륭수점총극륭수적비례래평개효모쌍잡교문고적질량.용PCR종Helicobacter pylor기인조중확증출p-37기인장기극륭입pGBKT7재체병사삽입편단여GAL4 BD동광,삽입편단적대소화서렬적정학성유쌍매절화측서득이증실.결과문고중공유1.9×106개독립극륭,함삽입편단극륭소점비례위91.7%.쌍매절화측서결과표명p37적정학삽입,Western blot 결과증명료융합단백적표체.결론구건료교고질량적cDNA문고화여GAL4 BD융합적유이단백,위진일보적연구전정료기출.
To construct the human gastric mucosa cDNA library and the recombinant plasmid pGBKT7-p37, the total RNA was isolated from normal gastric mucosa taken from surgically resected stomach of patient with gastric adenocarcinoma using TRIzol reagent under the direction of the manufacturer, and the double-strand cDNA was obtained through LD-PCR. Construction of cDNA library was performed by means of the direction of MATCHMAKER Library Construction and Screening Kit Clontech), and the quality of the two-hybrid library was checked by estimating the number of the independent clones obtained and the percentage of library clones containing an insert. The p37 gene was amplified from Helicobacter pylori genomic DNA and cloned in frame into vector pGBKT7. It was found that there were 1.9×106 independent clones in the library and the percentage of insert-containing clones was 91.7%. As confirmed by the result of sequencing, the p37 gene had been inserted correctly into vector pGBKT7 and the expression of the fusion protein was confirmed by Western blotting analysis. Our results indicate that a high quality yeast two-hybrid cDNA library and a bait fused to GAL4 BD domain were constructed, thus providing foundation for further studies to find the proteins interacting with VacA.
