猪囊尾蚴T24基因的克隆、表达与免疫原性分析.pdf

摘 要 扩增出716bp的片段，扩增产物连接到pGEM．T 98％和100％。 I和SalI酶切， 将重组质粒pGEM—T24和原核表达载体pGEX．4T_1分别用EcoR 与GST形成融合形式，分子量大小为40ku左右，能被猪囊尾蚴阳性血清所识别；将表 组融合蛋白产物发生免疫学反应。 I和Not I酶切，亚克 将重组质粒pGEM．T24和真核表达载体pPIC9K分别用EcoR pPIC9K—T24。用Sal 采用G418浓度梯度法对所有在营养缺陷型选择培养基MD上生长出的转化子进行筛 选，获得的高拷贝重组菌株。用PCR检测表明有目的基因整合。用1％甲醇诱导表达重 毕赤酵母中成功表达，表达产物的分子量为21ku左右，能被人囊虫阳性血清所识别。 本研究克隆了猪囊尾蚴T24基因，分别在原核和真核表达系统中进行了表达，证实 其表达产物具有免疫原性，从而为研制猪囊尾蚴基因工程疫苗提供了理论依据和实验材 料。 关键词：猪囊尾蚴，T24基因，原核表达，真核表达，免疫原性 Abstract TotalRNAwasextractedfrom T24 were cellulosae，the designed Cysticercus genespecificprimers softwareA716 was RT-PCRand into amplifiedby ligatedpGEM-T usingOligo bpspecificfragment was thatthe vectorIt identifiedrestrictionendonuclease and analysis，PCR fragment Easy by sequencing contained theT24 The ofthenucleotide the completeopen frame(oRy)ofgene homologies reading and aminoacid were98％and1 withthe sequenceencoding sequence 00％respectivelycomparedsequence inGenebank published withEcoR theT24 Therecombinant was Iand plasmidpGEM—T24digested Sail，releasinggene The WasinsertedintotheEcoR1andNot1sitesofthe fragmentfragmentsubsequen