水疱性口炎病毒N基因的克隆及在原核中的高效表达
摘要
本论文由两部分构成，一是利用RT-PCR技术克隆出水疱性口炎病毒（VSV）的N基因，二是利用大肠杆菌原核表达系统，通过基因重组技术高效表达出N基因。主要实验和结果如下：
1.以VSV鸡胚呈纤维细胞毒为材料，参照已发表的VSV基因组序列，设计1对引物，采用反转录PCR(RT-PCR)扩增出VSV N基因，将之连接于克隆载体pGEM-T载体后,转化感受态细胞DH5a,经37℃过夜培养，挑取阳性克隆，将PCR和酶切鉴定为阳性的重组质粒测序,比较测序株，IND wild株，tsG31株，HR株相应核苷酸序列和氨基酸序列同源性，其核苷酸同源性高达99%；氨基酸同源高达99%，进一步证实了VSV N基因的高度保守性。
2.设计一对分别带有EcoRⅠ和XhoⅠ酶切位点的表达引物，从以上实验构建的重组质粒中扩增出VSV的N基因，分别与克隆载体pGEM-T Vector连接并转化，阳性克隆扩增后提取重组质粒pGEM-T-N1。EcoRⅠ和XhoⅠ双酶切pGEM-T-N1以及表达载体pET-30c(+)，以产生粘性末端。纯化回收后，将之与pET-30c(+)连接，并分别转化大肠杆菌BL21(DE3),获得重组质粒pETVSV-N。PCR、酶切和序列分析鉴定目的基因的插入位置、方向和读码框完全正确，1.0 mmol/L IPTG诱导得到分子量为53 kDa的目的蛋白，Western blot检测表明，表达的目的蛋白能被VSV阳性血清所识别，从而为建立用重组N蛋白作为诊断抗原的ELISA方法及其它血清学方法奠定了基础。
The paper has two parts. One is nucleotide sequencing of N gene of the VSV, the other is the expression of the N gene of the VSV in prokaryotic vector. The results are:
1. One pair of primers was designed according to the published sequences of VSV. The N gene of it were amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The amplified N fragments of the VSV was 1 300 bp in length by agarose gel electrophoresis. Then the fragment was cloned into the pGEM-T vector and was sequenced. Comparison on the nucleotide sequences and deduced amino acid sequences of the N gene IND wild strain、tsG31strain and HR strain showed that the homology of nucleotide sequence was 99%,and that of the amino acid sequence was 99%.These prove again that the N gene is highly conserved in VSV.
2. Another pair of expression primers with digestion site EcoR I and Xho I on were designed to amplify the N genes of the VSV by PCR on the basis of the recombinant plasmid constructed in the former experiment. The recombinant plasmids pETVSV-N were obtained after being cloned pGEM-T-N1 into expression vector pET-30c(+). The insert position, the orientation and the ORF were right by PCR,digestion and sequence analysis. The 53 kDa target proteins were produced by inducing with 1.0 mmol/L IPTG. Western-blot showed that the expressed proteins could be recognized by VSV positive serum. Thus, the recombinant N protein makes the basis for establishing ELISA method and other serum method .
1.以VSV鸡胚呈纤维细胞毒为材料，参照已发表的VSV基因组序列，设计1对引物，采用反转录PCR(RT-PCR)扩增出VSV N基因，将之连接于克隆载体pGEM-T载体后,转化感受态细胞DH5a,经37℃过夜培养，挑取阳性克隆，将PCR和酶切鉴定为阳性的重组质粒测序,比较测序株，IND wild株，tsG31株，HR株相应核苷酸序列和氨基酸序列同源性，其核苷酸同源性高达99%；氨基酸同源高达99%，进一步证实了VSV N基因的高度保守性。
2.设计一对分别带有EcoRⅠ和XhoⅠ酶切位点的表达引物，从以上实验构建的重组质粒中扩增出VSV的N基因，分别与克隆载体pGEM-T Vector连接并转化，阳性克隆扩增后提取重组质粒pGEM-T-N1。EcoRⅠ和XhoⅠ双酶切pGEM-T-N1以及表达载体pET-30c(+)，以产生粘性末端。纯化回收后，将之与pET-30c(+)连接，并分别转化大肠杆菌BL21(DE3),获得重组质粒pETVSV-N。PCR、酶切和序列分析鉴定目的基因的插入位置、方向和读码框完全正确，1.0 mmol/L IPTG诱导得到分子量为53 kDa的目的蛋白，Western blot检测表明，表达的目的蛋白能被VSV阳性血清所识别，从而为建立用重组N蛋白作为诊断抗原的ELISA方法及其它血清学方法奠定了基础。
The paper has two parts. One is nucleotide sequencing of N gene of the VSV, the other is the expression of the N gene of the VSV in prokaryotic vector. The results are:
1. One pair of primers was designed according to the published sequences of VSV. The N gene of it were amplified by Reverse Transcription Polymerase Chain Reaction (RT-PCR). The amplified N fragments of the VSV was 1 300 bp in length by agarose gel electrophoresis. Then the fragment was cloned into the pGEM-T vector and was sequenced. Comparison on the nucleotide sequences and deduced amino acid sequences of the N gene IND wild strain、tsG31strain and HR strain showed that the homology of nucleotide sequence was 99%,and that of the amino acid sequence was 99%.These prove again that the N gene is highly conserved in VSV.
2. Another pair of expression primers with digestion site EcoR I and Xho I on were designed to amplify the N genes of the VSV by PCR on the basis of the recombinant plasmid constructed in the former experiment. The recombinant plasmids pETVSV-N were obtained after being cloned pGEM-T-N1 into expression vector pET-30c(+). The insert position, the orientation and the ORF were right by PCR,digestion and sequence analysis. The 53 kDa target proteins were produced by inducing with 1.0 mmol/L IPTG. Western-blot showed that the expressed proteins could be recognized by VSV positive serum. Thus, the recombinant N protein makes the basis for establishing ELISA method and other serum method .
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