【作者】 康桂英；

【导师】 秦建华；

【作者基本信息】 河北农业大学 ， 预防兽医学， 2005， 硕士

【摘要】 本试验将堆型艾美耳球虫通过卵囊的纯化、研磨、胰酶脱囊、DEAE-52层析柱纯化、反复冻融、超声裂解制备出子孢子抗原,利用此抗原检测杂交瘤细胞上清液,获得阳性杂交瘤细胞。利用酸性酚-硫氰酸胍-氯仿法提取杂交瘤细胞总RNA,琼脂糖凝胶电泳证明鸡堆型艾美耳球虫特异性杂交瘤细胞基因组RNA提取成功。以提取的RNA为模板,利用引物Oligo（dT）18合成cDNA。根据Genebank上的相关序列,合成两对引物,并以cDNA为模板,扩增出轻链可变区和重链可变区。经琼脂糖检测,发现轻链可变区基因的大小约312bp,重链可变区基因在381bp左右。从琼脂糖凝胶上切下所需DNA片段,用玻璃奶回收试剂盒回收轻链、重链可变区,经琼脂糖凝胶电泳检测回收产物,将回收效果好的产物与克隆载体pMD18-T连接,转化于用氯化钙制备的感受态细胞JM109,蓝白斑法筛选出阳性重组子。以RV-M/M13-47为引物对阳性重组子进行鉴定,菌落-PCR检测证明八个阳性重组子均为阳性。提取阳性质粒DNA并测序。测序结果显示轻链可变区和重链可变区基因符合鸡抗体轻链可变区和重链可变区基因特征,但发现与抗原表位互补结合的互补决定区（CDR）基因有多数发生改变,且在轻链可变区基因的CDR3中有基因缺失现象。回收的轻链可变区和重链可变区基因产物等摩尔浓度混合后,用重叠延伸拼接法扩增出单链抗体基因,再用带限制性内切酶位点的引物（Nco Ⅰ和HindⅡ）扩增出带有特定酶切位点的单链抗体基因片段。Nco Ⅰ和HindⅢ双酶切单链抗体与表达载体pET-22b（+）,回收试剂盒回收酶切后的单链抗体与表达载体pET-22b（+）基因片段,连接试剂盒将单链抗体与表达载体pET-22b（+）基因片段连接,并将连接产物转化感受态细胞JM109。氨苄青霉素筛选阳性重组子,以T7promoter prime/T7terminator为引物进行菌落PCR,测定正确后,37℃摇菌扩增,测菌浓度至OD₆₀₀为0.6时,加入IPTG诱导表达,对表达产物进行SDS-PAGE初步检测。SDS-PAGE检测显示其相对分子量是31KD,与预期结果一致,证明单链抗体基因成功的在大肠杆菌中得到表达。更多还原

【Abstract】 E.acervulina sporozoites were prepared by incubating sporulated oocysts in trypsin and taurodeoxycholic acid in PBS in incubator. Sporozoites were harvested by centrifugation and purified from cellular debris on diethylaminoethyl cellulose column (DE52).Pelleted sporozoites in phosphate-buffered saline were disrupted by 3 freeze-thaw cycles,warmed to room temperature, sonicated on ice in PBS. The positive hybridoma was choiced by antigens.Total RNA was extracted from chicken hybridoma cell secreting MAbs against E.acervulina.The frist chain of cDNA which derived from the total RNA with Oligo(dT)18Primer by RT was used as template,VH and VL genes was amplified by PCR using gene specific primers. PCR pruduct be examined by agarose gel electrophoresis and purified by agarose gel DNA purificatin kit ,the result shows the genes of VH and VL amplified were about 381bp and 312bp respectively.The purified pruduct was cloned pMD18-T vector.The cloned plasmids were transformed into JM109.Colonies were selected on ampicillin-containing agar plates and plasmids were isolated from several independent clones.the specific recombinant plasmid was identified with RV-M primer and M13-47 primer by colony PCR. The positive cloned were sequenced by Sanger’s dideoxy sequencing method. Nucleotide sequence of the VH and VL genes sequenced were compared with the published VH1-JH and Vλ1-Jλsequence from the CB strain chicken.As expected, sequence difference between VH/VL and corresponding germline genes were identified, predominantly localized in complementary determining regions.in particular, the VL gene contained a nucleotide deletion in CDR3.same quality of purified VH and VLgenes were jointed together with a linker DNA by splicing overlap extension PCR and a desired fragment(scfv) was generated.After scfv and expression vector were digested with the corresponding restriction enzymes Nco I and Hindlll and purified on an agarose gel,the scfv gene was ligated into expression vector pET-22b(+)and transformed into competent E.coli JM109 cells.the recombinants was grown on the ampr plate. The aimed was identified with T7 promoter primer and T7 terminator primer by colony PCR.the result indicated that the resultant construct contained the gene of scfv fragment at right orientation.Fermentation of the positive coined was更多还原