目的:构建人类8型疱疹病毒(HHV-8)小衣壳蛋白(ORF65)基因重组原核表达质粒并诱导表达,获得其重组融合蛋白。方法:软件设计引物,在引物两端添加特异性酶切位点,以pGEM-Teasy/ORF65为模板,PCR扩增基因序列,克隆入原核表达载体pET-41a中,...目的:构建人类8型疱疹病毒(HHV-8)小衣壳蛋白(ORF65)基因重组原核表达质粒并诱导表达,获得其重组融合蛋白。方法:软件设计引物,在引物两端添加特异性酶切位点,以pGEM-Teasy/ORF65为模板,PCR扩增基因序列,克隆入原核表达载体pET-41a中,构建原核表达质粒pET41a-ORF65,转化大肠杆菌(E.coli)DH5x,经酶切、测序鉴定其插入序列的正确性,转入E.coli BL21(DE3),并诱导表达,采用聚丙烯酰胺凝胶电泳(SDS-PAGE)方法以及序列分析软件DNAMAN 5.0、Vector NTI Suite 6、Primer Primier 5对表达蛋白进行生物信息学分析。结果:测序结果表明所构建pET41a-ORF65原核表达质粒连接正确,SDS-PAGE表明ORF65基因获得成功表达,生物信息学分析显示该蛋白含有1个N糖基化位点,2个蛋白激酶C磷酸化位点,4个胆碱激酶Ⅱ磷酸化位点,4个N肉豆蔻酰化位点,1个酰胺化位点。BlastP数据库的相似性检索发现有与之有较高同源性的蛋白。结论:成功表达HHV-8病毒小衣壳蛋白ORF65,该蛋白与疱疹病毒衣壳蛋白的同源性较高,是疱疹病毒衣壳蛋白家族中的一员。展开更多
To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic...To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.展开更多
The C12orf65 gene is a nuclear gene that encodes a mitochondrial matrix protein contributing to mitochondrial translation. C12orf65 gene-related diseases are rare and present with large heterophenotypes. Most of the r...The C12orf65 gene is a nuclear gene that encodes a mitochondrial matrix protein contributing to mitochondrial translation. C12orf65 gene-related diseases are rare and present with large heterophenotypes. Most of the reported patients have had optic atrophy with intellectual disability, encephalomyopathy, spastic paraplegia, and ophthalmoplegia. Peripheral neuropathy has been reported in one thmily. Here, we report a case of a Chinese patient with optic atrophy and distal motor neuropathy due to a novel compound heterozygous mutation in the C12orf65 gene.展开更多
文摘目的:构建人类8型疱疹病毒(HHV-8)小衣壳蛋白(ORF65)基因重组原核表达质粒并诱导表达,获得其重组融合蛋白。方法:软件设计引物,在引物两端添加特异性酶切位点,以pGEM-Teasy/ORF65为模板,PCR扩增基因序列,克隆入原核表达载体pET-41a中,构建原核表达质粒pET41a-ORF65,转化大肠杆菌(E.coli)DH5x,经酶切、测序鉴定其插入序列的正确性,转入E.coli BL21(DE3),并诱导表达,采用聚丙烯酰胺凝胶电泳(SDS-PAGE)方法以及序列分析软件DNAMAN 5.0、Vector NTI Suite 6、Primer Primier 5对表达蛋白进行生物信息学分析。结果:测序结果表明所构建pET41a-ORF65原核表达质粒连接正确,SDS-PAGE表明ORF65基因获得成功表达,生物信息学分析显示该蛋白含有1个N糖基化位点,2个蛋白激酶C磷酸化位点,4个胆碱激酶Ⅱ磷酸化位点,4个N肉豆蔻酰化位点,1个酰胺化位点。BlastP数据库的相似性检索发现有与之有较高同源性的蛋白。结论:成功表达HHV-8病毒小衣壳蛋白ORF65,该蛋白与疱疹病毒衣壳蛋白的同源性较高,是疱疹病毒衣壳蛋白家族中的一员。
基金Knowledge Innovation Program of the Chinese Academy of Sciences(0702121Y-J1)
文摘To purify the protein encoding the small capsid protein (SCP) of KSHV and analyze its immunogenicity, the carboxyl terminus of orf65 of Kaposi's sarcoma associated-herpesvirus (KSHV) was expressed in a prokaryotic expression system. The expression of recombinant E.coli containing pQE-80L-orf65 was induced by isopropyl-β-D-thiogalactopyranoside (IPTG) and the fusion protein was purified by chromatography. The expressed protein and its purified product were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and showed that 9 kDa was the expected size of the purified orf65 protein. The antiserum was produced in rabbit which was immunized by purified orf65 protein. An ELISA assay was established to analyze the immunogenicity of the purified orf65 protein. The ELISA analysis demonstrated that orf65 protein has strong immune activity, and the immune activity of polyclonal antibody against orf65 was more than 4 fold higher than that in the serum of the non-immunized rabbit. These results demonstrate that purified orf65 protein has very strong immunogenicity and can be used in screening KSHV infection in the general population using ELISA.
基金This study was supported by a grant of the National Natural Science Foundation of China （No. 81471185）.Acknowledgments We thank the patient and her family for their participation in this study. We also thank the MyGenome Institute for their kind administrative assistance.
文摘The C12orf65 gene is a nuclear gene that encodes a mitochondrial matrix protein contributing to mitochondrial translation. C12orf65 gene-related diseases are rare and present with large heterophenotypes. Most of the reported patients have had optic atrophy with intellectual disability, encephalomyopathy, spastic paraplegia, and ophthalmoplegia. Peripheral neuropathy has been reported in one thmily. Here, we report a case of a Chinese patient with optic atrophy and distal motor neuropathy due to a novel compound heterozygous mutation in the C12orf65 gene.
