Isolation and preparation of potentilla anserina galactoside reference substance 蕨麻苷对照品的分离制备
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Malvidin - 3 - o - galactoside chloride 氯化锦葵色素
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In this paper , first strand cdna of 3abc gene was synthesized using template rna extracted from cells infected with fmdv . the complete 3abc gene about isoobp was amplified by pcr and ligated into pgem - t easy vector . after transforming e . coli dh5 a , ampicillin resistant colonies were isolated and plasmid dna was prepared and analyzed by restriction analysis and pcr . presence of the full length 3abc gene was verified by nucleotide sequence analysis and the plasmid containing the expected sequence was named as pgem - 3abc . comparing the aquired sequence of 3abc with that of reference strains , the homology is more than 99 percent . the pgem - 3abc was digested with sal i and bgl ii and ligated into xho i and bgl ii - digested expression vector ptriex - 4 neo . lt was identified by restriction analysis and pcr and sequencing that this fragment had a 17bp deletion hi the nucleotide sequence 708bp of 3abc gene , which happened to form a terminator codon behind 3ab gene , but it contained the complete open reading frame ( orf ) of 3ab gene . positive clones were selected and induced with lmmol / l isopropyl - d - galactoside ( iptg ) , bacteria were detected by sds - page and western blotting after properly treated . the results showed that the 3ab gene expressed successfully in e . coli and 33 . 5ku fusion protein can be recognized by the positive bovine serum of fmdv . the amount of target protein is over 26 % of the total bacteria protein by gel thin layer scanning analysis 扩增产物连接到pgem - teasy载体中，转化大肠杆菌dh5菌株，筛选氨苄青霉素抗性菌落，提取质粒经酶切鉴定、 pcr分析以及确证性测序证明，所克隆的1500bp左右的片段含有完整的3abc基因，与国外参考序列相比，同源性在99以上。将重组质粒pgem - 3abc和表达载体ptriex - 4neo分别用sal和bgl与xho和bgl消化后，亚克隆3abc基因至原核表达载体ptriex - 4neo中，通过酶切鉴定、 pcr扩增以及序列分析，发现克隆到ptriex - 4neo载体上的片段于3abc基因708bp处出现了17bp的缺失，碰巧在3ab基因后形成一终止密码子，但3ab基因的阅读框架完整，选出含有3ab基因完整阅读框架的阳性克隆，用iptg诱导表达，收集菌液进行sds - page电泳、 westernblotting分析，结果表明， 3ab基因在大肠杆菌中成功表达，其表达产物为分子量33 . 5ku的融合蛋白，并能被口蹄疫病毒阳性血清识别。经薄层扫描分析，表达量占总蛋白量的26以上。
