摘要
目的：为了研究人ｅｒａ基因这一人类新基因表达产物的组织分布情况．方法：根据人ｅｒａ序列设计并合成扩增引物，用ＰＣＲ法从ｐＵＣ１９－ｈｅｒａ质粒中扩增人ｅｒａ基因（ｈ－ｅｒａ）全长ｃＤＮＡ和ｈ－ＥＲＡＣ端的结构域基因，并分别克隆到（Ｈｉｓ）６融合表达载体ｐＲＳＥＴ－Ｃ和非融合表达载体ｐＤＨ中，诱导表达（ＨＩＳ）６－ｈ－ＥＲＡ 融合蛋白和 ｈ－ＥＲＡＣ端结构域蛋白．用 ｈ－ＥＲＡＣ端结构域蛋白免疫新西兰白兔，制备兔抗 ｈ－ＥＲＡ抗血清．用 Ｗｅｓｔｅｒｎ－ｂｌｏｔ对兔抗ｈ－ＥＲＡ 抗血清．进行了鉴定，并成功地制备了兔抗ｈ－ＥＲＡ血清．利用在大肠杆菌中高表达、纯化的（Ｈｉｓ）６－ｈ－ＥＲＡ融合蛋白，对已制备的兔抗人 ＥＲＡ抗血清进行进一步鉴定纯化获得抗 ｈ－ＥＲＡ的特异性多抗；再对 ４月～５月人胎儿心、肝、肺、脾、肾等脏器和胃、小肠、睾丸、胸腺、胰脏及胆囊进行免疫组织化学染色．结果：在人类４月～５个月胎儿心、肝、肺、脾、肾脏等脏器和胃、小肠、睾丸中均有免疫反应阳性产物出现．胰脏、心、肺脏等是高表达，脾脏、胃、小肠是中等表达，睾丸、肝脏、肾脏呈低表达，胆囊、胸腺无免疫阳性表达产物出现．结论：人 ＥＲＡ在人类正常胎儿不同组织中?
Objective: To observe the distribution of human new gene-era in human fetal different tissues by using expressed protein of h-era and h-era C-terminal domain in E.coli and prepared polycloning antiserum against human era. Method: Gene clone, PCR, protein expressing, purifying, Western-blot identify and immunohistochemistry staining were used. Human era (h-era) cDNA, h-era and C-terminal domain gene were amplified by PCR using a plasmid containing h-era c-DNA as a template, E. colt TAP106 transformed with the recombinant plasmid pDH-h-era was introduced at 42 C to express h-era C-terminal domain protein. Antiserum against h-era was prepared by immunizing rabbit with h-era C-terminal domain protein purified by SDS-PAGE. E. colt BL21 (DE3) transformed with the recombinant plasmid pRSET-C-h-era was induced with IPTG to express (His) 6-h-era fusion protein that was applied for Western-blot analysis to identify the antiserum. Finally using the characteristic antibody to Immunohistochemical stain human fetal tissue. Results: The expressed h-era C-terminal domain and (his) 6-h-era fusion protein were about 40% and 80% of total bacteria protein respectively. The new hera protein had different expressing level by using the purifying characteristic polycloning antibody in fetal different tissues in our study. Conclusion f The h-era C-terminal domain protein and (His) 6-h-era fusion protein were highly expressed in E.coli. and the rabbit antiserum against h-era was successfully prepared and characterized. The h-era protein might be a new functional protein and it could express in most normal human fetal tissues.
出处
《伤残医学杂志》
2001年第2期3-6,共4页
Medical Journal of Trauma and Disability
