大鼠RVLG cDNA的克隆、原核表达和小鼠抗RVLG蛋白多克隆抗体的制备

Cloning and Prokaryotic Expression of Rat RVLG and Preparation of Mouse Anti-RVLG Polyclonal Antibody

摘要:

为了识别大鼠卵巢中的生殖细胞, 我们在原核系统中表达和纯化RVLG蛋白并制备了多克隆抗体。采用RT-PCR方法从大鼠睾丸组织中扩增获得RVLG cDNA片段, 然后克隆到pMD19-T载体上进行测序, 经双酶切回收目的基因片段后, 将其插入到原核表达载体pGEX-4T-1上, 转入大肠杆菌BL21(DE3)中诱导表达。纯化后的GST-RVLG融合蛋白免疫昆明(KM)小鼠, 最后给小鼠腹腔注射S180细胞制备抗RVLG腹水多克隆抗体。用Western blotting及免疫组织化学法鉴定RVLG腹水多克隆抗体的特异性, 间接ELISA法测定该抗体的效价。序列分析表明, 我们所克隆的RVLG cDNA片段比GenBank中报道的大鼠RVLG cDNA(NM_001077647)多60 bp, 原因是由于RVLG的可变剪切方式造成的。本文成功构建了重组表达质粒pGEX-RVLG, 且GST- RVLG融合蛋白在大肠杆菌BL21(DE3)中高效表达, 表达的目的蛋白占菌体总蛋白的10%以上。制备的抗体可特异性识别RVLG蛋白, 其效价达1:20 000。获得的高效价、高特异性的小鼠抗RVLG蛋白腹水多克隆抗体为下阶段研究RVLG的特异性表达奠定了基础。

Abstract:

In order to identify rat ovarian germ cells, we expressed and purified rat RVLG protein in Escherichia coli cells and prepared a mouse anti-rat RVLG polyclonal antibody. The rat RVLG cDNA was obtained from rat testicle tissue by RT-PCR and was cloned into the vector pMD19-T. Sequence analysis proves that the cloned RVLG cDNA fragment was 60 bp longer than that released in the GenBank (NM_001077647), resulting from an alternative splicing of the RVLG pre-mRNA. The RVLG cDNA was double digested with the restriction endonucleases BamH I and EcoR I, and then was extracted from gel and inserted into the prokaryotic expression vector pGEX-4T-1. The recombinant expression plasmid pGEX-RVLG was verified for successful construction and then was transformed into Escherichia coli BL21(DE3) for induction to express the GST-RVLG fusion protein by IPTG. The GST-RVLG fusion protein was expressed in Escherichia coli BL21 (DE3) at a high level which accounts for more than 10% of the total bacterial cellular protein. The purified RVLG protein was used as an antigen to immunize KM mouse for the production of polyclonal antibody in ascetic fluid followed by celiacly injecting the mouse with S180 cells. The mouse anti-rat RVLG antibody was analyzed by ELISA, Western blotting and immunohistochemistry for its specificity and titer. The antibody could recognize RVLG protein specifically and its titer was about 1:20 000. These results confirm that the mouse anti-rat RVLG polyclonal antibody with high affinity and specificity has been prepared successfully, and lay a foundation for our ongoing research on the specific expression of RVLG in rat ovary.