CAJ | 학술논문

为了对微小隐孢子虫（C ． p arv um）类钙调蛋白（CM L ）基因进行原核表达，分析重组表达蛋白的反应原性。以 C ．parvum卵囊cDNA为模板，用PCR方法扩增得到C ．parvum CML基因。将CML基因连接到克隆载体pMD18‐T ，获得重组质粒pMD‐CML ，经限制性内切酶 BamH Ⅰ和 Xho Ⅰ双酶切后，连接到经相同内切酶酶切的表达载体pGEX‐6p‐1上，构建重组表达质粒，转化到大肠埃希菌BL21（DE3）中进行诱导表达。利用GST 亲和树脂法纯化重组表达蛋白，对纯化的重组蛋白进行Western blot分析。结果表明，成功构建了重组原核表达质粒pGEX‐CML ，重组质粒转化菌经IPTG诱导后成功地表达出了分子质量约为51 ku的重组蛋白rCM L ，纯化的蛋白rCM L能与感染兔隐孢子虫（C ．cuniculus）的兔血清发生特异性反应，具有很好的反应原性。
위료대미소은포자충（C ． p arv um）류개조단백（CM L ）기인진행원핵표체，분석중조표체단백적반응원성。이 C ．parvum란낭cDNA위모판，용PCR방법확증득도C ．parvum CML기인。장CML기인련접도극륭재체pMD18‐T ，획득중조질립pMD‐CML ，경한제성내절매 BamH Ⅰ화 Xho Ⅰ쌍매절후，련접도경상동내절매매절적표체재체pGEX‐6p‐1상，구건중조표체질립，전화도대장애희균BL21（DE3）중진행유도표체。이용GST 친화수지법순화중조표체단백，대순화적중조단백진행Western blot분석。결과표명，성공구건료중조원핵표체질립pGEX‐CML ，중조질립전화균경IPTG유도후성공지표체출료분자질량약위51 ku적중조단백rCM L ，순화적단백rCM L능여감염토은포자충（C ．cuniculus）적토혈청발생특이성반응，구유흔호적반응원성。
In order to express Cryptosporidium parvum calmodulin‐like protein (CML) gene in E .coli BL21(DE3) and analyze the antigenicity of the recombinant protein ,CML gene was amplified by PCR with cDNA of C .parvum oocysts .The amplified CML gene was cloned into pMD18‐T vector and the DNA of recombinant pMD‐CML plasmid was extracted .The plasmid was digested with double enzymes and the ob‐jective fragments were connected with pGEX‐6p‐1 which had been digested with same enzymes .After iden‐tifying by double restrict enzyme digestion and gene sequence analysis , the recombinant plasmids were transformed to E .coli BL21(DE3) cells and the transformed bacteria was induced to express with IPTG . Recombinant proteins were purified by High‐Affinity GST · Bind Resin affinity chromatography .Antigen‐icity of the recombinant proteins was analyzed by Western blot .The results showed that the prokaryotic expression vector pGEX‐CML was constructed successfully and an approximate 51 ku recombinant protein rCML was expressed successfully after inducing with IPTG .The purified recombinant protein could be recognized specifically by the sera from rabbit infected with C .cuniculus .