鹅干扰素-α在毕赤酵母中的分泌表达及抗病毒活性研究
摘要
干扰素(interferons, IFNs)是一种可诱导细胞因子的多基因家族,其主要作用是抵抗病毒感染的免疫防御以及抗增殖和免疫调节作用,作为生物药剂和疫苗佐剂具有广阔的应用前景。
为了更好的探讨基因工程技术来开发重组鹅IFN-α制剂的可能性,本研究将去除信号肽的鹅α干扰素基因置于毕赤酵母α因子分泌信号肽后,构建能分泌表达goIFN-α蛋白的重组表达质粒pPICZaA-goIFN-α,将其转化到毕赤酵母宿主菌X33中,实现了其在毕赤酵母中的高效表达。并采用微量细胞病变抑制法分析研究了goIFN-α生物活性,为进一步的研究与开发鹅干扰素类制剂打下基础。其主要结果如下：
1、根据NCBI上本课题组已提交的天府肉鹅基因序列(序列号为HQ115583),设计并合成一对引物,应用PCR技术从含有天府肉鹅IFN-α完整基因的重组质粒pMD18-T-goIFN-α中扩增鹅α干扰素的成熟肽基因。PCR产物经纯化回收后,克隆到pMD18-T载体上。将DNA序列测定正确的克隆质粒pMDT-IFNa用EcoRⅠ和XbaⅠ双酶切后与同样经EcoRⅠ和XbcⅠ双酶切的pPICZαA连接过夜,转化大肠杆菌感受态DH5a,得到重组表达质粒pPICZaA-goIFN-a,并对重组表达质粒进行PCR鉴定、酶切鉴定以及序列测定。
2.将验证正确的重组表达质粒用sacⅠ进行单酶切,采用电转化法将线性化的重组质粒电穿孔导入毕赤酵母宿主菌X33,涂布YPDS+Zeocin选择性培养基,置30。C温箱培养3-5天,结果获得多个白色的菌落。分别挑选生长良好的3株不同的白色单菌落,先用BMGY培养基激活培养,离心后用BMMY培养基重悬菌体,每隔24小时添加1%的甲醇进行诱导表达72小时后,离心收集菌液。冻干法浓缩后的菌液,经斑点杂交筛选出高拷贝的转化子。将筛选出的高拷贝菌落重新进行诱导表达,并用Elisa法确定最佳的表达时间和甲醇浓度。重组子经最佳表达方法诱导表达后,离心收集菌液,用TCA浓缩后的蛋白,进行SDS-PAGE和Western-blotting分析,结果表明分泌于胞外的鹅IFN-α蛋白分子量大约为22 kDa,比理论值(18.7 kDa)略大,估计是表达产物在表达过程中发生一定的糖基化所致。
3.采用微量细胞病变抑制法评价了表达的重组鹅IFN-α的抗病毒活性。制备鹅胚成纤维细胞,采用TCID50法测定VSV病毒株的鹅胚成纤维细胞的半数感染量。表达的蛋白经透析、过滤除菌处理后,分析研究其生物活性,结果显示鹅IFN-α对水泡型口炎病毒在鹅胚成纤维细胞中可起抑制作用,抗病毒活性为1.79×103U/ml。
综上所述,本研究成功的克隆了天府肉鹅干扰素-α基因,成功构建了鹅IFN-α的真核表达质粒,实现了其在毕赤酵母中的高效表达,表达产物并具有明显的抗病毒活性。
Interferons (interferons, IFNs) are a multigene family of inducible cytokines which mediate antiviral, immunomodulatory, and antiproliferative effects. IFNs have broad applied prospects as biological agents and vaccine asjuvants.
In order to discuss the possibility of recombinant goose interferon-a, in this study, the signal peptide gene of goIFN-a was removed and then was placed into the pichia pastorisα-factor secretion signal sequence to reconstruct the recombinant expression plasmid pPICZaA-goIFN-a. Then the recombinant plasmid was transformed into pichia pastoris host strain X33, and realized its high efficient expression. And its antiviral activity was evaluated by using VSV on goose embryo fibroblast, which provides a good foundation for the research and development of goose interferon. The main results of this reseatrch are as followed:
1. The primers were designed and synthesized by the gene sequence of TianFun goose IFN-a, which had been submitted into NCBI (HQ115583). Then the goose IFN-a gene of mature peptide was amplified from the recombinant plasmid pMD 18-T-goIFN-a containing the complete sequence of Tianfu goose IFN-a by PCR, afer the PCR product was purified and recycled, it was colned into pMD18-T vector. After DNA sequencing was correct, the recombinant plasmids pMDT-IFNa was digested by EcoR I and Xba I, and ligated with pichia pastoris shuttle vector pPICZaA which was also digested by EcoR I and Xba I. Then the ligated liquid was transformed into E.coli DH5a to construct the recombinant expression plasmids pPICZaA-goIFN-a. The recombinant plasmids were suffered by PCR, restrict enzyme digestion and sequence analysis.
2.The correct recombinant expression plasmid was digested by sac I and the linearized recombinant plasmid was transformed into pichia pichia strain X33 by electroporation. Putting them on the selective YPDS culture medium containing zeocin at 30℃for 3-5 days, and then many white transformants would been screened on the culture medium. Three different white single clonies was pick up and induced first by BMGY culture medium, and then using BMMY culture medium to resuspended the cell. After the culture medium was induced by adding 1% methanol every 24h for 72h, the culture was centrifugated and collected. The multi-copy recombinants were picked by dot blot hybridization, and then it was reinduced to express the protein, and then the expression conditions of the best time and methanol concentration were optimized by Elisa. After the recombinants were expressed by the best methods, it was concentrated by TCA and was deteced by SDS-PAGE and Western-blotting assays. The SDS-PAGE and Western-blotting assays demonstrated that tf-goIFNα, about a 22 kDa protein, was successfully secreted into the culture medium, it was a little bigger that the predicted (18.7 kDa), it was estimated that the expression products was glycosylated durning the expression.
3. The antiviral activity was evaluated by using VSV on goose embryo fibroblast. The goose embryo fibroblast cells were preparated, and its half amount of infected with VSV was determinated by TCID50.Then after the protein was dialyzed and filtration sterilized, its antiviral activity was evaluated, and the rusults revealed that goose interferon-a could inhibit the VSV on goose embryo fibroblast and had an antiviral activity of 1.79×103U/ml.
In summary, this study has successfully cloned goose interferon-a gene, and successfully constructed the eukaryotic expression plasmid of goIFN-a, achieved its highly expressed in pichia pastoris and the expression products have significant biological activity.
为了更好的探讨基因工程技术来开发重组鹅IFN-α制剂的可能性,本研究将去除信号肽的鹅α干扰素基因置于毕赤酵母α因子分泌信号肽后,构建能分泌表达goIFN-α蛋白的重组表达质粒pPICZaA-goIFN-α,将其转化到毕赤酵母宿主菌X33中,实现了其在毕赤酵母中的高效表达。并采用微量细胞病变抑制法分析研究了goIFN-α生物活性,为进一步的研究与开发鹅干扰素类制剂打下基础。其主要结果如下：
1、根据NCBI上本课题组已提交的天府肉鹅基因序列(序列号为HQ115583),设计并合成一对引物,应用PCR技术从含有天府肉鹅IFN-α完整基因的重组质粒pMD18-T-goIFN-α中扩增鹅α干扰素的成熟肽基因。PCR产物经纯化回收后,克隆到pMD18-T载体上。将DNA序列测定正确的克隆质粒pMDT-IFNa用EcoRⅠ和XbaⅠ双酶切后与同样经EcoRⅠ和XbcⅠ双酶切的pPICZαA连接过夜,转化大肠杆菌感受态DH5a,得到重组表达质粒pPICZaA-goIFN-a,并对重组表达质粒进行PCR鉴定、酶切鉴定以及序列测定。
2.将验证正确的重组表达质粒用sacⅠ进行单酶切,采用电转化法将线性化的重组质粒电穿孔导入毕赤酵母宿主菌X33,涂布YPDS+Zeocin选择性培养基,置30。C温箱培养3-5天,结果获得多个白色的菌落。分别挑选生长良好的3株不同的白色单菌落,先用BMGY培养基激活培养,离心后用BMMY培养基重悬菌体,每隔24小时添加1%的甲醇进行诱导表达72小时后,离心收集菌液。冻干法浓缩后的菌液,经斑点杂交筛选出高拷贝的转化子。将筛选出的高拷贝菌落重新进行诱导表达,并用Elisa法确定最佳的表达时间和甲醇浓度。重组子经最佳表达方法诱导表达后,离心收集菌液,用TCA浓缩后的蛋白,进行SDS-PAGE和Western-blotting分析,结果表明分泌于胞外的鹅IFN-α蛋白分子量大约为22 kDa,比理论值(18.7 kDa)略大,估计是表达产物在表达过程中发生一定的糖基化所致。
3.采用微量细胞病变抑制法评价了表达的重组鹅IFN-α的抗病毒活性。制备鹅胚成纤维细胞,采用TCID50法测定VSV病毒株的鹅胚成纤维细胞的半数感染量。表达的蛋白经透析、过滤除菌处理后,分析研究其生物活性,结果显示鹅IFN-α对水泡型口炎病毒在鹅胚成纤维细胞中可起抑制作用,抗病毒活性为1.79×103U/ml。
综上所述,本研究成功的克隆了天府肉鹅干扰素-α基因,成功构建了鹅IFN-α的真核表达质粒,实现了其在毕赤酵母中的高效表达,表达产物并具有明显的抗病毒活性。
Interferons (interferons, IFNs) are a multigene family of inducible cytokines which mediate antiviral, immunomodulatory, and antiproliferative effects. IFNs have broad applied prospects as biological agents and vaccine asjuvants.
In order to discuss the possibility of recombinant goose interferon-a, in this study, the signal peptide gene of goIFN-a was removed and then was placed into the pichia pastorisα-factor secretion signal sequence to reconstruct the recombinant expression plasmid pPICZaA-goIFN-a. Then the recombinant plasmid was transformed into pichia pastoris host strain X33, and realized its high efficient expression. And its antiviral activity was evaluated by using VSV on goose embryo fibroblast, which provides a good foundation for the research and development of goose interferon. The main results of this reseatrch are as followed:
1. The primers were designed and synthesized by the gene sequence of TianFun goose IFN-a, which had been submitted into NCBI (HQ115583). Then the goose IFN-a gene of mature peptide was amplified from the recombinant plasmid pMD 18-T-goIFN-a containing the complete sequence of Tianfu goose IFN-a by PCR, afer the PCR product was purified and recycled, it was colned into pMD18-T vector. After DNA sequencing was correct, the recombinant plasmids pMDT-IFNa was digested by EcoR I and Xba I, and ligated with pichia pastoris shuttle vector pPICZaA which was also digested by EcoR I and Xba I. Then the ligated liquid was transformed into E.coli DH5a to construct the recombinant expression plasmids pPICZaA-goIFN-a. The recombinant plasmids were suffered by PCR, restrict enzyme digestion and sequence analysis.
2.The correct recombinant expression plasmid was digested by sac I and the linearized recombinant plasmid was transformed into pichia pichia strain X33 by electroporation. Putting them on the selective YPDS culture medium containing zeocin at 30℃for 3-5 days, and then many white transformants would been screened on the culture medium. Three different white single clonies was pick up and induced first by BMGY culture medium, and then using BMMY culture medium to resuspended the cell. After the culture medium was induced by adding 1% methanol every 24h for 72h, the culture was centrifugated and collected. The multi-copy recombinants were picked by dot blot hybridization, and then it was reinduced to express the protein, and then the expression conditions of the best time and methanol concentration were optimized by Elisa. After the recombinants were expressed by the best methods, it was concentrated by TCA and was deteced by SDS-PAGE and Western-blotting assays. The SDS-PAGE and Western-blotting assays demonstrated that tf-goIFNα, about a 22 kDa protein, was successfully secreted into the culture medium, it was a little bigger that the predicted (18.7 kDa), it was estimated that the expression products was glycosylated durning the expression.
3. The antiviral activity was evaluated by using VSV on goose embryo fibroblast. The goose embryo fibroblast cells were preparated, and its half amount of infected with VSV was determinated by TCID50.Then after the protein was dialyzed and filtration sterilized, its antiviral activity was evaluated, and the rusults revealed that goose interferon-a could inhibit the VSV on goose embryo fibroblast and had an antiviral activity of 1.79×103U/ml.
In summary, this study has successfully cloned goose interferon-a gene, and successfully constructed the eukaryotic expression plasmid of goIFN-a, achieved its highly expressed in pichia pastoris and the expression products have significant biological activity.
引文
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