摘要
目的:制备生长激素释放激素受体剪接变异体1(GHRHR-SV1)抗体,并检测GHRHR-SV1蛋白在黑色素瘤细胞中的表达情况,为后续的肿瘤机制研究奠定基础。方法:设计GHRHR-SV1基因序列,将其插入质粒pET-32a(+)中,构建pET-GHRHR-SV1重组载体,经PCR及测序鉴定后,转入大肠杆菌BL21(DE3)中,分别加入0、0.5、1.0、1.5和2.0mmol·L^(-1)异丙基-β-D-硫代半乳糖苷(IPTG);诱导1、2、4和6h,并在37℃、30℃和25℃不同温度下进行诱导;观察目的蛋白表达的最佳IPTG浓度、时间和温度。亲和层析纯化重组蛋白,将纯化后的蛋白免疫家兔,制备兔多克隆抗体,ELISA法检测该抗体效价,Western blotting法检测GHRHR-SV1蛋白在黑色素瘤细胞系B16-F10中的表达情况。结果:重组载体pET-GHRHR-SV1构建成功。在IPTG浓度为2.0mmol·L^(-1)时目的蛋白的表达量最高;IPTG诱导蛋白表达2h时,蛋白表达量最大;当温度为25℃时,蛋白的表达量最大。诱导表达的蛋白相对分子质量约为24 000,纯化后纯度为80%,GHRHR-SV1抗体效价为1∶1 600 000,B16-F10细胞中有GHRHR-SV1蛋白表达。结论:成功制备pETGHRHR-SV1重组载体。GHRHR-SV1蛋白在IPTG浓度为2.0mmol·L^(-1)、诱导时间为2h和温度为25℃的条件下表达最佳。成功制备了高效价的GHRHR-SV1抗体。黑色素瘤细胞系中有GHRHR-SV1蛋白表达。
Objective：To prepare the polyclonal antibody against growth hormone-releasing hormone receptor splice variant 1（GHRHR-SV1）,and to detect the expressions of GHRHR-SV1 protein in melanoma cells,and to lay a foundation for further study of the pathogenesis of tumor.Methods：The gene sequence of GHRHR-SV1 was designed and synthesized,and then cloned into pET-32a（＋）plasmid to construct the recombinant vector pETGHRHR-SV1.After identification by PCR and sequencing,the recombinant plasmid was transformed into E.coli BL21（DE3）.Then 0,0.5,1.0,1.5,and 2.0mmol·L^（-1） isopropylβ-D-1-thiogalactopyranoside（IPTG）were added respectively to induce for 1,2,4,and 6hat 37℃,30℃,and 25℃.The optimal dosage,time and temperature of IPTG of the target protein were observed.The protein was expressed under the optimal condition and purified by affinity chromatography.The polyclonal antibody was obtained by immunizing the rabbit with the purified protein.The titer was assayed by ELISA method.The expression of GHRHR-SV1 protein in B16-F10 cells was detected by Western blotting method.Results： The recombinant vector pET-GHRHR-SV1 was constructed successfully.The expression amount of GHRHR-SV1 protein was the highest when it was induced by2.0mmol·L^（-1） IPTG for 2hat 25℃.The relative molecular weight of induced protein was 24 000.After purification,the purity of protein was 80%.The GHRHR-SV1 antibody titer was 1∶1 600 000.The GHRHRSV1 protein was expressed in B16-F10 cells.Conclusion：The recombinant vector pET-GHRHR-SV1 is constructed successfully.The expression amount of GHRHR-SV1 protein in the condition of 2.0 mmol·L^（-1） IPTG,2hinduction and at 25℃is the highest.The high titer of ployclonal antibody is obtained.The GHRHR-SV1 protein can express in melanoma cells.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2016年第6期1237-1242,共6页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金资助课题(31271476)
广东省广州市科技局科技计划项目资助课题(20150010076)
广东省教育厅人才项目资助课题(2013KJCX0155)
广东省自然科学基金资助课题(2016A030313601)
关键词
生长激素释放激素受体剪接变异体1
重组载体
重组蛋白
抗体
黑色素瘤
growth hormone-releasing hormone receptor splice variant 1
recombinant vector
recombinant protein
antibody
melanoma
