恒河猴CGβ亚基(rmCGβ)DNA在HeLa细胞中的表达及其对小鼠免疫反应的诱导

EXPRESSION OF BETA SUBUNIT OF RHESUS MONKEY CHORIONIC GONADOTROPIN (rmCGβ) DNA IN HeLa CELLS AND THE IMMUNE RESPONSES IN BALB/C MOUSE INOCULATED BY rmCGβ DNA VACCINE

为了探讨人绒毛膜促性腺激素hCGβ亚基DNA避孕疫苗的可能性 ,以恒河猴绒毛膜促性腺激素rmCGβ亚基全长氨基酸编码序列的cDNA片段构建了真核质粒表达载体pCMV4 rmCGβ。通过脂质体转染的方法导入HeLa细胞 ,采用RT PCR方法检测rmCGβ在mRNA水平上的表达。实验证明HeLa细胞在转染后 2 4h表达最强 ,48h和 72h表达依次减弱 ;利用ELISA方法检测了 pCMV4 rmCGβ在HeLa细胞中rmCGβ蛋白的表达 ,rmCGβ蛋白主要以细胞内或膜结合的形式存在 ,其含量可达 0 5 μg/ 10 6细胞。pCMV4 rmCGβDNA免疫接种雌性BALB/c纯系小鼠 ,能诱导小鼠产生强烈的体液免疫应答 ,滴度最高可达 1∶6 0 0 0以上 ,高滴度的免疫反应持续 10周以上 ,且免疫反应的强度与pCMV4 rmCGβDNA无剂量依赖关系。

The human chorionic gonadotropin (hCG) as one of glycoprotein hormones family shares a common alpha subunit but differs in the hormone-specific beta subunit. Because of the physiological and temporal specificity, the hCGβ is a good target molecular to develop immuno-contraceptives. To study the feasibility of human DNA immuno-contraceptive by using hCGβ gene, the Rhesus monkey was chosen as an in vitro and in vivo model to understand the immune response, anti-fertility effect and safety of β-CG DNA immuno-contraceptive. pCMV4-rmCGβ, a kind of DNA vaccine inserted with full-coding cDNA sequence of beta subunit of rhesus monkey(Macaca mulatta) chorionic gonadotropin (rmCGβ), was constructed to understand the feasibility of developing human DNA immuno-contraceptive. In this work, the full-coding cDNA sequence of rmCGβ was inserted into eukaryotic expression vector pCMV4 to construct recombinant expression vector pCMV4-rmCGβ. Using HeLa cells transient expression system, the capability of expression of pCMV4-rmCGβ was detected in vitro, in which the mRNA and the protein of rmCGβ expressed in HeLa cells were determined by RT-PCR and ELISA, respectively. The pCMV4-rmCGβ DNA were transfected into HeLa cells with lipid, and the culture sera and cells were collected at 24 h, 48 h, and 72 h after transfection. The results showed that there was the highest level expression of rmCGβ at 24 h after transfection at mRNA level, and the expression was gradually decreased from 48 h to 72 h. The mRNA expression of treatment assay showed significant difference compare to untreated HeLa cells and Lipofectamine Lipid control assay (P<0.05). At protein level, the highest level expression of rmCGβ reached 0.5 μg (equivalent to hCGβ)/10 6 cells, and the rmCGβ protein was mainly with intracellular or membrane associated state. To detect the ability of biological function of pCMV4-rmCGβ plasmid DNA in vivo, the female BALB/c mice were inoculated with pCMV4-rmCGβ plasmid DNA from 10 μg to 100 μg with intramuscular inoculation method. The sera samples were collected at 2 week, 4 week, 6 week, and 10 week post-immunization. The results of ELISA showed that the intensive humoral immune responses in female BALB/c mice were elicited by inoculating pCMV4-rmCGβ DNA vaccine. The highest level of antibody titer was above 6 000. These intensive immune responses were no dose-dependent on the quantity of pCMV4-rmCGβ DNA from 10 μg to 100 μg, and could last at least 10 weeks. The results indicate that the recombinant pCMV4-rmCGβ eukaryotic expression vector has biological function, which can express rmCG β-subunit in vitro cells and in vivo tissue. Moreover, the rmCGβ protein could be expressed by the muscle of BALB/c mice and presented by the Antigen Present Cells to induce intensive humoral immune responses. In brief, This work establishes the base for further anti-fertility and safety detection of pCMV4-rmCGβ DNA vaccine, and should be valuable in developing CGβ DNA immuno-contraceptive.