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目的构建羊型布氏菌脂多糖O-侧链合成必需的甲酰基转移酶WbkC基因真核表达质粒,并进行鉴定。方法提取16M株羊型布氏菌基因组DNA,PCR扩增WbkC基因,克隆至psos载体上,构建诱饵重组质粒psos-WbkC,转化酵母菌株cdc25Hα,进行菌落PCR鉴定,并检测其在酵母双杂交系统中的自激活和定位情况。结果诱饵重组质粒psos-WbkC经PCR双酶切及测序证明构建正确;重组酵母菌经PCR鉴定,可扩增出约780 bp的目的基因;诱饵质粒在酵母双杂交系统中无自激活且定位正确。结论已构建了羊型布氏菌WbkC基因真核表达质粒psos-WbkC,可应用于酵母双杂交系统中,用于寻找巨噬细胞cDNA文库中与WbkC相互作用的蛋白质。
Objective To construct eukaryotic expression plasmid of formyltransferase WbkC, which is necessary for the O-side chain of LPS lipopolysaccharide, and to identify it. Methods The genomic DNA of 16M Streptococcus mutans was extracted, and the WbkC gene was amplified by PCR. The bait plasmid was cloned into psos vector. The bait recombinant plasmid psos-WbkC was constructed and transformed into yeast strain cdc25Hα. The PCR products were identified by PCR. In the self-activation and positioning of the situation. Results The bait recombinant plasmid psos-WbkC was confirmed by PCR double-restriction enzyme digestion and sequencing. The recombinant yeast was identified by PCR and amplified about 780 bp. The bait plasmid had no self-activation and was correctly located in the yeast two-hybrid system . Conclusion The eukaryotic expression plasmid psos-WbkC of WbkC gene of B. lamblia has been constructed and can be used in yeast two-hybrid system to find the protein interacting with WbkC in macrophage cDNA library.