摘要
旋毛虫肌幼虫排泄－分泌（ＥＳ）抗原来源于体外培养的旋毛虫肌幼虫的排泄－分泌物。为研制具有ＥＳ抗原功能的旋毛虫基因重组抗原，首先用ＡＧＰＣ法提取旋毛虫肌幼虫总ＲＮＡ并进行变性琼脂糖凝胶电泳分析，发现它缺少真核生物所具有的２８ＳｒＲＮＡ。根据编码４５０００和４９０００蛋白的基因结构，设计并合成２对ＰＣＲ引物，用ＲＴ－ＰＣＲ方法分别扩增出了２个目的基因（ＴＳＰＧ和ＴＳＰＧⅡ）。序列分析发现，ＴＳＰＧ在第４５８到５０５位之间的核苷酸编码框架与结构基因Ｐ４５的不同，此外，还有多处出现与Ｐ４５不同的碱基。在ＴＳＰＧⅡ核苷酸序列中只有１个碱基与Ｐ４９不同。根据可识别的糖基化位点判定，ＴＳＰＧ和ＴＳＰＧⅡ各含有１个Ｎ－型连接的糖基化位点。将构建的３个重组质粒转化大肠杆菌，通过温度诱导培养，分别在大肠杆菌中表达出３７０００、４６０００和３４０００的重组蛋白，三者的表达水平分别为２２％、１０％和８％；Ｗｅｓｔｅｒｎｂｌｏｔ分析和ＥＬＩＳＡ检测表明，它们均可被猪旋毛虫病阳性血清和单克隆抗体识别，但特异性有所不同。将ＴＳＰＧⅡ克隆至Ｅ．ｃｏｌｉ－Ｙｅａｓｔ穿梭载体ｐＨＩＬ－Ｓ１上，经内切酶消化和Ｓｏｕｔｈｅｒｎｂｌｏｔ杂交鉴定后，?
The ES antigen of T.spiralis was derived from excretorysecretory products of T.spiralis muscle larvae incubated in vitro. In order to develop an ESlike gene recombinant antigen, the total RNA of T.spiralis was extracted with improved AGPC technique. Analyzed by denaturation agarose gel electrophoresis, it was found that the total RNA of T.spiralis had no 28S rRNA which exists in most of the eukaryotic cells. The two PCR primers were designed and synthesized on the basis of the known sequences and two structural genes (TSPG and TSPG Ⅱ) encoding 45 000 and 49 000 proteins were obtained by using RTPCR technique. By sequencing, the reading frame of the deduced amino acid sequence between the positions 458 and 505 base in the TSPG nucleotide sequence was quite different with that in the P45 sequence, which encodes the different amino acid sequence (16 amino acids). Moreover, there were many onebase change in TSPG sequence as compared with the known sequence. The nucleotide sequence of TSPGⅡ was almost the same with the P49 sequence except one base. According to the recognized glycosylation sites, there was one Nlinked glycosylation site in each sequence. The recombinant plasmids were transformed and the three recombinant proteins (37 000, 46 000 and 34 000) expressed in E.coli by temperatureinduced culture with the expression levels being 22%, 10% and 8%, respectively. The expressed proteins were recognized in Western blot analysis and ELISA by sera from swine infected with T.spiralis and McAb against T.spiralis, but they showed slightly different specificity. The TSPGⅡgene was cloned into the E.coliyeast shuttle vector pHILS1 and then transformed into yeast (GS115). After methanolinduced incubation, a 45 000 recombinant protein was found in the supernatant of the culture and the expression level peaked at the time of 72 h post incubation (1.05 g/L). The expressed protein could be recognized by positive sera from swine. Based
出处
《中国兽医学报》
CAS
CSCD
北大核心
1997年第6期581-588,共8页
Chinese Journal of Veterinary Science
关键词
旋毛虫
ES抗原
基因克隆
高效表达
Trichinella spiralis
ES antigen
gene cloning
high level expression
