The Construction Of Transgenic Mice Carring HIL-10 And EGFP Gene

Human interleukin-10 (hIL-10) which is secreted by T lymphocytes,monocytes, macrophages B lymphocytes and other cells is the unique cytokines with the function of downregulating immunity. It plays an immunosuppressive,immunomodulatory and anti-inflammatory role mainly by regulating immune cells’action.In clinical,it is widely used in the treatment of many diseases such as inflammatory bowel disease, acute lung injury, rheumatoid arthritis, hepatitis C, reperfusion injury, psoriasis, multiple sclerosis, experimental sepsis shock and so on.Mammary gland bioreactor is a Transgenic technology that use milk protein gene to construct mammary specific expression vector,which can direct the target gene express in in the mammary gland of transgenic animals efficiently and specificly.Through this technology foreign protein with biological activity can be gained from the milk of transgenic animals.Mammary gland is an specialized endocrine gland which can produce pharmaceutical proteins with full biological activity. Mammary gland bioreactorhas unparalleled advantages in the production of pharmaceutical proteins,And for its high biological activity of protein production, purification simple, low investment, low cost, no pollution, and so on.it is called "Molecules farm".In this study,by PCR technology,bovine β-casein (bβ-CN) gene 5 ’regulatory region sequence was cloned from cattle genomic DNA;HIL-10 gene coding sequence and its upstream regulatory sequences,part of intron sequences was cloned from human genomic DNA.And the two are connected by DNA ligase. EGFP promoter and the coding sequence was obtained from the pEGFP-N1 plasmidin the method of double digestion.And then,they were injected into the male pronuclears of mouse’s zygotes through co-injection.After in vitro culture,morphologically normal embryos were transplanted into the uterus of the recipient mice.The results as follows:1.Cloning of hIL-10:According to the sequence of X78437 on the GeneBank, A pair of primers were designed and synthesized. The sequence consisting of 1558bp was obtained by PCR, Which was linked with pEGM-T.After identified by PCR and digested by enzyme. The sequencing shew the obtained DNA was homologous to X78437 by 99%. hIL-10 gene was cloned successfully.2.Cloning of bovine β-CN gene 5 ’regulatory region:A pair of primers were designed and synthesized based on the gene sequence of bβ-CN on the GeneBank (X14711).The sequence consisting of 745bp was obtained by PCR, Which was linked with pEGM-T.After identified by PCR and digested by enzyme. The sequencing indicted the target DNA was homogous to X14711 by 98.29%.Bovine β-CN gene 5 ’regulatory regiongene was cloned successfully.3.The connection of the target gene:The 5’end of bβ-CN regulatory sequences and hIL-10 gene were connected by DNA ligase,and then 2303bp DNA sequence was obtained.The sequence was linked with pEGM-T, After identified by PCR and digested by enzyme. The sequencing indicted the ligation product was homogous to target genes by 98.19%.The target genes were linked successfully.4.EGFP promoter region and the entire coding sequence 1900bp in tatol were obtained from plasmid pEGFP-N1 by double digestion.5.Microinjection and embryo transfer:After microscopic injection and in vitro culture 300 normal 2-cell embryos were transplanted to the oviduct of 25 pregnant female rats.But no pregnancy reaction were found;all pregnant female rats in the expected date of birth were anatomied,but no stillbirth were found.