猕猴溶组织内阿米巴原虫病病原分离鉴定及凝集素hgl基因的原核表达
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摘要
本研究从猕猴阿米巴病例的腹泻粪样中分离到一株阿米巴原虫,该原虫经过实验室的培养和形态学观察后,初步鉴定为溶组织内阿米巴或迪斯帕内阿米巴,采用鉴别溶组织内阿米巴和迪斯帕内阿米巴的虫种特异性引物,对阿米巴原虫peroxiredoxin基因进行PCR扩增,结果显示溶组织内阿米巴特异性引物扩增出了相应大小的DNA片断,经过克隆后测序,序列分析(登录号为:EU022309)显示本虫株与人溶组织内阿米巴株序列同源性为98%—99%,而与人迪斯帕内阿米巴株序列同源性仅为89%,鉴定该阿米巴猕猴分离株为溶组织内阿米巴,命名为Entamoeba histolytica MH530株。
参照GenBank中收录的人溶组织内阿米巴原虫半乳糖/乙酰氨基半乳糖凝集素的重链hgl基因序列设计一对引物,通过PCR扩增获得与预期相符的产物,经过克隆后测序,得到1761bp的有效基因序列。通过对该基因核苷酸序列和推导的氨基酸进行分析,结果表明核苷酸序列与人溶组织内阿米巴的序列同源性在93.44%-96.21%之间,与人迪斯帕内阿米巴的序列同源性为87.44%。该基因编码了一个有587个AA的多肽,分子量为65.514KD,等电点理论值为5.17,具有较强的亲水性,蛋白抗原性分析显示其抗原位点分布于整个蛋白质多肽链。该序列已登录在GenBank上(登录号为EU244479)。
根据获得的基因序列设计一对表达引物,对其中重要的含多个抗原决定簇的LC3段进行原核表达,制备目的基因和表达载体,将目的片断插入pET32 b(+)表达载体上,将鉴定正确的重组质粒转化大肠杆菌BL21感受态细胞,用1.0mM/L的IPTG诱导表达了猕猴溶组织内阿米巴凝集素hgl基因LC3段,SDS—PAGE分析表明,在相对分子质量约66KD处出有明显的蛋白质表达条带,而对照组pET32 b(+)空质粒菌在此处没有条带,该蛋白在3h时基本达到表达量最大值,而IPTG浓度在0.05mM/L和2.0mM/L之间没有明显表达差异,均都得到了很好的表达。
An Entamoeba sp.strain was isolated from an adult female rhesus macaque with symptom including dysentery and diarrhea.Through observing the morphous and measuring the sizes of amoeba trophozoites and cysts,we diagnosed the isolated strain as E.histolytica/E,dispar.Using the species-specific primers for E.histolytica and E.dispar (p11 plus p12 and p13 plus p14,respectively)for PCR diagnosis,we diagnosed this case as E.histolytica natural infecting in rhesus macaque.
Base on the published nucleotide sequence of human E.histolytica hgl gene,a pair of PCR primers was designed and synthesized.Total genome DNA isolated from the Entamoeba sp.strain was used as template to PCR amplification.The PCR production was cloned into PMD18-T vector,and 1761bp DNA fragment was obtained by sequencing.By blasting the homologous sequences in Genbank databases,DNA sequence was confirmed as the E.histolytica hgl gene sequence.The determined nucleotide sequences were compared pair wise for similarity,the results showed that the sequence of hgl gene from rhesus macaque E.histolytica was between 93.44%-96.21%percent identify to the human E.histolytica hgl genes,was 87.44%percent identify to the human E.Dispar hgl gene.This DNA sequence encodes a protein consisting of 587 amino acid(AA).The molecular mass of polypeptide was 65.514KD,its isoelectric point was 5.17,also has the strong hydrophilicity and antigenicity.The sequence produced in this study was deposited in Genbank(Accession No.EU244479).
By the technology of DNA recombination,the LC3 segment of hgl gene was cloned into expression plasmid pET-32b(+),and was transformed to E.coli BL21.The recombinant bacteria were induced to expression through the changes of IPTG.The results showed that a new protein band was found in SDS-PAGE with molecular mass of about 66KDa,which was consisted of a 46KDa protein of the hgl gene and pET-32b(+)(20KDa). The amount of proteinum was to peak at 3h after IPTG induced.There were almost no difference to amount of proteinum in different IPTG concentration,all be high.
参照GenBank中收录的人溶组织内阿米巴原虫半乳糖/乙酰氨基半乳糖凝集素的重链hgl基因序列设计一对引物,通过PCR扩增获得与预期相符的产物,经过克隆后测序,得到1761bp的有效基因序列。通过对该基因核苷酸序列和推导的氨基酸进行分析,结果表明核苷酸序列与人溶组织内阿米巴的序列同源性在93.44%-96.21%之间,与人迪斯帕内阿米巴的序列同源性为87.44%。该基因编码了一个有587个AA的多肽,分子量为65.514KD,等电点理论值为5.17,具有较强的亲水性,蛋白抗原性分析显示其抗原位点分布于整个蛋白质多肽链。该序列已登录在GenBank上(登录号为EU244479)。
根据获得的基因序列设计一对表达引物,对其中重要的含多个抗原决定簇的LC3段进行原核表达,制备目的基因和表达载体,将目的片断插入pET32 b(+)表达载体上,将鉴定正确的重组质粒转化大肠杆菌BL21感受态细胞,用1.0mM/L的IPTG诱导表达了猕猴溶组织内阿米巴凝集素hgl基因LC3段,SDS—PAGE分析表明,在相对分子质量约66KD处出有明显的蛋白质表达条带,而对照组pET32 b(+)空质粒菌在此处没有条带,该蛋白在3h时基本达到表达量最大值,而IPTG浓度在0.05mM/L和2.0mM/L之间没有明显表达差异,均都得到了很好的表达。
An Entamoeba sp.strain was isolated from an adult female rhesus macaque with symptom including dysentery and diarrhea.Through observing the morphous and measuring the sizes of amoeba trophozoites and cysts,we diagnosed the isolated strain as E.histolytica/E,dispar.Using the species-specific primers for E.histolytica and E.dispar (p11 plus p12 and p13 plus p14,respectively)for PCR diagnosis,we diagnosed this case as E.histolytica natural infecting in rhesus macaque.
Base on the published nucleotide sequence of human E.histolytica hgl gene,a pair of PCR primers was designed and synthesized.Total genome DNA isolated from the Entamoeba sp.strain was used as template to PCR amplification.The PCR production was cloned into PMD18-T vector,and 1761bp DNA fragment was obtained by sequencing.By blasting the homologous sequences in Genbank databases,DNA sequence was confirmed as the E.histolytica hgl gene sequence.The determined nucleotide sequences were compared pair wise for similarity,the results showed that the sequence of hgl gene from rhesus macaque E.histolytica was between 93.44%-96.21%percent identify to the human E.histolytica hgl genes,was 87.44%percent identify to the human E.Dispar hgl gene.This DNA sequence encodes a protein consisting of 587 amino acid(AA).The molecular mass of polypeptide was 65.514KD,its isoelectric point was 5.17,also has the strong hydrophilicity and antigenicity.The sequence produced in this study was deposited in Genbank(Accession No.EU244479).
By the technology of DNA recombination,the LC3 segment of hgl gene was cloned into expression plasmid pET-32b(+),and was transformed to E.coli BL21.The recombinant bacteria were induced to expression through the changes of IPTG.The results showed that a new protein band was found in SDS-PAGE with molecular mass of about 66KDa,which was consisted of a 46KDa protein of the hgl gene and pET-32b(+)(20KDa). The amount of proteinum was to peak at 3h after IPTG induced.There were almost no difference to amount of proteinum in different IPTG concentration,all be high.
引文
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