Transient Expression Of The Human Interleukin-2 (Ser-125) In Milk Of Rabbits And Goats

Interleukin-2 (IL-2) is a cytokine secreted by activated T lymphocytes and has an important role in a number of in vivo immune responses. The important function of IL-2 is to induce T cell proliferation and differentiation and to promote helper T cells and NK cells to product cytokines. Therefore, IL-2 is used as anticancer and imrnunotherapy agent in clinic because of its extensive biological activity. Nature IL-2 is consist of 133 amino acid, and has 3 cysteine in 58, 105 and 158 site, it's necessary to maintain biological activity of IL-2 that a disulfide bond formed by Cys-58 and Cys-105. It's easy to form mismatching or dimmer and reduce activity because of Cys-125. Therefore, it can dispel the molecular basis for forming mismatching and dimmer and makes purification simple that Cys-125 of nature IL-2 substituted by Ser-125.Mammary land bioreactor of animal is recognized increasingly for its low cost and high yield. It's a long-period and high-expense work to establish a mammary land bioreactor; the foreign gene will be expressed stochastically in the tested animal. Therefore, it's necessary to verify the feasibility of vectors before establishing large transgenic animal.In this study, Cys-125 of nature HuIL-2 was substituted by the Ser-125 using PCR for site-directed mutation, and the 867bp 5' flanking sequence of goat β -lactoglobulin(BLG) gene was isolated from fresh blood of goat by PCR method. One mammary-specific expression vectors of pBLG- Ser-125HuIL-2 were constructed with BLG gene promoter and cDNA of human interleukin-2 (Ser-125). The vectors mixed with liposome were injected into mammary glands of later stage of pregnant rabbits and goats through mammary gland center duct. Milk was collected on l-10d from rabbits and goats after parturition, and Ser-125HuIL-2 in themilk was detected by ELISA assays. Six rabbits were tested, one was injected control, another was injected pIL-2 with liposome, two were injected expression vectors, another two were injected expression vectors with liposome. The detection results showed that the rabbits which were injected control and pIL-2 didn't express Ser-125HuIL-2, one of the two rabbits which was injected vectors directly expressed Ser-125HuIL-2, and the expression level of Ser-125HuIL-2 were at the range of 0.06-0.53ug/L in the milk, but another didn't express Ser-125HuIL-2, the rate of success was 50%. Two rabbits that were injected expression vectors with liposome expressed Ser-125HuIL-2, the rate of success was 100%, and the expression levels of Ser-125HuIL-2 were at the range of 0.18-2.36ug/L in the milk. Three goats were tested, one was injected control, two were injected expression vectors with liposome. The detection results showed that two goats which were injected expression vectors with liposome expressed Ser-125HuIL-2, the rate of success was 100%, the expression level of Ser-125HuIL-2 were at the range of 0.83-6.45|ig/L in the milk. This study expressed Ser-125HuIL-2 successfully using mammary land of rabbits and goats in normal physiological condition, and simplified the method of verification the feasibility of mammary-specific expression vectors, and founded a firm foundation to establishing large transgenic animal.