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皮蝇Hypodermin C和Hypodermin A基因的克隆及在大肠杆菌中的表达
Cloning of Hypodermin C and Hypodermin A Gene of Hypoderma.sp and Expression in Escherichia.coli
【作者】 杨东英;
【作者基本信息】 西北农林科技大学 , 基础兽医学, 2003, 硕士
【摘要】 解决皮蝇蛆病的早期诊断和免疫防治问题意义重大,要得到及时有效的诊断和免疫需要大量的诊断和免疫试剂。本试验的目的旨在进行Hypodemin C (HC)和Hypodermin A (HA)基因的克隆、测序、构建重组表达载体并诱导表达,获得重组抗原,以解决天然抗原的不足并为诊断和免疫试剂的产业化奠定基础。 本试验从皮蝇二期幼虫中提取获得了总RNA,反转录为单链cDNA,用上下游引物扩增出了目的基因。扩增产物连接到PGEM-T easy载体上,转化入大肠杆菌JM109中进行蓝白斑筛选后,用酶切、PCR鉴定和测序的方法鉴定出重组阳性质粒(PGEM-HC和PGEM-HA)。随后将重组质粒PGEM-HC、PGEM-HA和表达载体PGEX-4T-1分别以相同的限制酶(BamH Ⅰ、Not Ⅰ酶切后,将酶切的目的片段(HC和HA)亚克隆到原核表达载体中构建重组表达载体(PGEX-4T-1-HC和PGEX-4T-1-HA)并转化到宿主菌BL21中。重组质粒酶切(BamH Ⅰ、Not Ⅰ)、PCR鉴定筛选出阳性克隆,测序证明目的基因正确插入到表达载体。用IPTG诱导表达,收集不同时间的菌液进行SDS-PAGE电泳、Western-blotting分析检测。 本试验可得出如下结论: 1.从皮蝇二期幼虫中提取获得总RNA,利用RT-PCR技术扩增获得了大小为713bp的Hypodermin C和大小为700bp的Hypodermin A基因。 2.分别将目的基因Hypodermin C和Hypodermin A与克隆载体(pGEM T easy)连接并转化到宿主菌JM109中,构建了重组克隆载体pGEM-HC和pGEM-HA。 3.序列分析表明,所克隆获得的基因与GenBank中已经登录的核苷酸和氨基酸的同源性分别为:HC98.4%和100%,HA97.2%和99.3%,证明本试验虫株与国外报道的同源性很高。 4.构建了两个原核表达载体PGEX-HC和PGEX-HA,重组载体含有包括起始密码子、前导信号肽序列和终止密码子在内的完整的开放阅读框架。 5.筛选出的阳性克隆,用IPTG诱导表达,表达产物通过SDS-PAGE分析,证明HypoderminC基因和HypoderminA基因在大肠杆菌中得到了表达,表达产物是分子量分别为54kD和56kD的融合蛋白。从天然皮蝇幼虫中提取了皮蝇抗原,通过SDS一PAGE分离获得了HyPoderminC和HyPoderminA天然抗原,用此抗原免疫家兔,制备了皮蝇HyPoderminC和HyPoderminA阳性血清。通过Westem·blotting分析表达产物,表明表达产物具有免疫活性,能被制备的阳性血清所识别。
【Abstract】 Total RNA was extracted from the second stage larve of Hypoderma sp, then single chain cDNA was synthesized by reverse transcription using oligo(dT)18 as a primer. The Hypodermin C (HC) and Hypodermin A (HA) gene specific primers were devised by DNASTAR software. 713bp and 700bp specific fragments were amplified by PCR and ligated into PGEM-T easy vector. It was identified by restriction endonuclease digest analysis, PCR and sequencing that this fragment contained the complete open reading frame (ORF) of the HC and HA gene. After restriction digest the PGEM-HC, PGEM-HA and the PGEX-4T-1, subcloned the interested genes of HC and HA into the prokaryotic expression vector PGEX-4T-1. Positive clones were selected by restriction endonuclease analysis and PCR identification.The expression was induced by IPTG, then the culture was collected in different times and tested by SDS-PAGE and Western-bloting. It showed that the HC and HA genes were expressed successfully in E.coli. The 54 kD and 56 kD fusion proteins can be recognized by the positive serum of Hypoderma.Conclusions can be deduced as follows :1. Total RNA was extracted from the second stage larve of Hypoderma sp, The 713bp of Hypodermin C (HC) and 700bp of Hypodermin A (HA) gene were amplified by RT-PCR2. Recombinant cloning vector of pGEM-HC and pGEM-HA were constructed by ligating the HC and HA genes into pGEM T east vector and transformating into JM109.3. In comparison with Genbank data,the homologies of the nucleotide sequence and amino acid sequence were as following: HC was 98.4% and 100%; HA was 97.2% and 99.3% respectively.4. Two recombinant prokaryotic expression vector were constructed which has complete open reading occlusing initation codon, leader signal peptide sequence and termination codon.5. The expression was induced by IPTG, then the culture was collected in different times and tested by SDS-PAGE. It showed that the HC and HA genes were expressed successfully in E.coli.6. HC and HA antigens were separated by SDS-PAGE from nature hypoderma sp larve,then immunized rabbitsrespectivelly, HC and HA positive serum were preparated.7. The expression products were tested by Wester-blotting, it demonstrated the fusion proteins can be recognized by positive serum of hypoderma sp.
【Key words】 Hypoderma; Hypodermin C; Hypodermin A; Cloning; Expression;
- 【网络出版投稿人】 西北农林科技大学 【网络出版年期】2004年 01期
- 【分类号】Q78
- 【下载频次】130