【作者】 陈俊锋；

【导师】 俞纯方； 黄兵；

【作者基本信息】 西南大学 ， 基础兽医学， 2006， 硕士

【摘要】 TA4基因是柔嫩艾美耳球虫（E．tenella）孢子化卵囊的一个表面抗原基因，其表达的蛋白具有一定的免疫原性；EtMIC₄是编码E．tenella细胞器微线蛋白的基因，在子孢子及裂殖子阶段表达，分泌的蛋白向后覆盖于虫体的表面，与宿主细胞表面相黏附，参与虫体的运动及入侵。本文以TA4基因和与球虫入侵宿主细胞有关的EtMIC₄部分基因作为研究目标，根据Genbank中登录的序列设计引物，利用RT-PCR技术从E．tenella（上海株）孢子化卵囊中扩增出TA4基因和EtMIC₄基因的一部分。经琼脂糖凝胶电泳检测，将大小与TA4基因预计分子量一致的片段纯化后连接到pGEM-T-easy克隆载体中，再转化大肠杆菌DH5a感受态细胞，得到的转化子经PCR鉴定和酶切分析筛选阳性克隆；将大小与EtMIC₄基因片段预计分子量一致的片段纯化并经EcoR Ⅰ和Hind Ⅲ双酶切回收后，与经EcoR Ⅰ和Hind Ⅲ双酶切回收的pET-28-a和pET-32-a表达载体连接。结果表明：①得到了含有TA4基因的阳性重组子（pGEM-TA4），经核苷酸测序，该序列全长773bp，其中TA4基因的阅读框为651bp，编码216个氨基酸，与国外报道的TA4基因（登录号：M21004.1）比较，序列的同源性为100％（773／773）。与国内报道的TA4（浙江株）基因（登录号：DQ327836.1）比较，序列的同源性为99％（765／770），即在TA4（浙江株）基因序列的38bp、51bp、229bp、462bp和635bp处的碱基分别是G、A、G、T、T而TA4（上海株）的则是A、C、A、A、C，其中51bp和229bp处碱基不同，其翻译后相应的氨基酸也不同，在浙江株是Met和Arg，而在上海株则是Leu和Lys，其它氨基酸相同：②得到了含有EtMIC₄基因片段的阳性重组子（pET-28-a-EtMIC₄和pET-32-a-EtMIC₄），经核苷酸测序，该基因序列全长1351bp，其中基因的阅读框为948bp，编码315个氨基酸，该序列与Tomley等发表的序列（登录号：AJ306453.1）进行网上比对后发现：两者之间有99％（1138／1140）的同源性，即在EIMIC₄（登录号：AJ306453.1）序列的6061bp和6126bp处的碱基分别为T和G，而在EtMIC₄（上海株）则是C和A；此外在EtMIC₄（登录号：AJ306453.1）序列的5510bp处比其多125个碱基，但多的碱基位于基因的阅读框之外；与杜爱芳等发表的5401基因序列（登录号：AY819649.1）有99％（861／864）同源性；推测这种碱基不同的现象，可能是由于E．tenella不同地理株之间存在一定的遗传差异所导致的。将经测序为正确的重组克隆质粒（pGEM-TA4）用EcoR Ⅰ和HindⅢ双酶切并回收后，与经EcoRⅠ和HindⅢ双酶切回收的pET-28-c相连接；用EcoR Ⅰ和Sal Ⅰ双酶切并回收后，与经EcoR Ⅰ和Sal Ⅰ双酶切回收的pGEX-4T-2相连接，转化大肠杆菌DH5a感受态细胞，得到的转化子经PCR鉴定和酶切分析，筛选出符合正确阅读框的重组子，构建成重组表达质粒（pET-28-c-TA4和pGEX-4T-2-TA4）。更多还原

【Abstract】 The TA4 gene and EtMIC₄ gene were cloned and sequenced, and its prokaryotic expression vectors were constructed to gain the genenal information for further studying recombinant vaccine against coccidiosis in this paper. According to the reporded sequences, two pair of primers were designed and synthesized, two genes from sporozoited oocysts of E.tenella （shanghai strain） were amplified by RT-PCR. The PCR products were checked by agrose gel electrophoresis and purified by agrose gel fraction method.The purified products of TA4 were ligated to pGEM-T-easy vector successfully. The ligated products were transferred into content cell DH5a. The specific recombinant plasmid was identified by PCR and restriction endonuclease analysis. The results indicated that the resultant construct contained the gene of interest TA4 at right orientation of the insert. The positive cloned was sequenced by Sanger’s dideoxy sequencing method. The results demonstrated the size of TA4 gene was 773 bp in length and included an open reading frame encoding a protein of 216 amino acid residues. The cDNA of TA4 （SH） was identical with TA4（M21004.1） and shared a high of 99% identity with that of reported TA4（ZJ）（DQ327836.1）. and at the 38 bp、51 bp、229 bp、462 bp and 635 bp of the sequence of TA4 gene, the bases mutated respectively from G、A、G、T、T into A、C、A、A、C, but the mutation did not rusult in change of open reading frame. The reasons given rise to reduce nucleotides may be there was a genetic diverse in different Eimeria strains. Furthermore, the fragment encoding the TA4 gene was excised from the positive clone pGEM-TA4 by EcoR I and Hind III and purified by agrose gel fraction method. Then the fragment was subcloned into the pET-28-c expression vector digested by EcoR I and Hind HI restriction enzymes; The fragment encoding the TA4 gene was excised from the positive clone pGEM-TA4 by EcoR I and Sal I and purified by agrose gel fraction method. Then the fragment was subcloned into the pGEX-4T-2 expression vector digested by EcoR I and Sal I restriction enzymes.The recombinant expression plasmids were identified by PCR and restriction enzymes analysis. The results indicated the fragment was correctly inserted into the pET-28-c and pGEX-4T-2 expression vector and conformed to a reading frame. The fusion protein were expressed in E.coli BL21 （DE3 ） host with a predicted molecular weight of 25 kDa and 53 kDa. The purified products of EtMIC₄ were digested by EcoR I and Hind HI, Then the fragments were ligated into the pET-28-a and pET-32-a expression vectors digested by EcoR I and Hind III restriction enzymes. The ligated products were transferred into content cell DH5a. The specific recombinant plasmid was identified by PCR and restriction endonuclease analysis. The positive cloned was sequenced by Sanger ’ s dideoxy sequencing method. The results demonstrated the size of EtMIC₄ gene was 1351 bp in length and included an open reading frame encoding a protein of 315 amino acid residues.. Compared with the EtMIC₄, EtMIC₄（SH） was inserted 125 bases at the 5510 bp of EtMIC₄.The reasons given rise to reduce nucleotides may be there was a genetic diverse in different Eimeria strains.更多还原