| Interleukin-10 (Interleukin-10, IL-10), as an important regulator of theimmune system factors, is mainly produced by Th2 cells,.It is the only cytokinereduced the role of immune cytokines, and has a wide range of biological activities,including immune suppression anti-inflammatory and immunomodulatoryproperties.The main function of IL-10 is reflecting the limitation and terminating theinflammation, regulating a variety of immune cell differentiation and proliferation, butalso has immune stimulating properties of conditioning, clear the infection and non-infectious particles. Studies have shown that human interleukin 10 IL-10 physsignificant roles in the processes of many diseases such as inflamnmtion,canceration,autoimmune diseases,tumor and grit rejection.IL-10 is widely used in clinical acutelung injury, inflammatory bowel disease, septic shock and experimental sepsis,psoriasis, rheumatoid arthritis, multiple sclerosis, hepatitis C, reperfusion injury andother diseases in clinical application.Animal mammary gland bioreactor was invented by Gordon in 1989, It makes theidio-expression of exogenous gene in animal mammary gland by transgenictechnology. This technology was widely used in the production of pharmaceuticalproteins.for its high biological activity of protein production, purification simple, lowinvestment, low cost, no pollution, and so on.In order to construct recombinant vector and mammary gland bioreactor thatstably express hIL-10 protein, In this study, bovineβ-casein (bβ-CN) gene 5'regulatory region sequence and hIL-10 gene were cloned from cattle, human genomicDNA. the recombinant vector pcDNA3.1 (+) -bβCN-hIL-10 were constructed by ligateβ-CN5' regulatory region and hIL-10 sequence into pcDNA3.1(+). Inject pcDNA3.1(+)-bβCN-hIL-10 plasmid,which was linearized by Bst1107 I single-digested, into theoriginal core of the fertilized egg by microinjection, then transfer the healthy embryosinto mouse uterus. The results are as follows:1.Cloning of hIL-10: According to the sequence of X78437 on theGeneBank(X78437 ), A pair of primers were designed and synthesized. The sequenceconsisting of 1993bp was obtained by PCR, Which was linked with pUM-T.Afteridentified by PCR and digested by enzyme. The sequencing shew the obtained DNAwas homologous to X78437 by 99.19% . hIL-10 gene was successful cloned.2.Cloning of bovineβ-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 3165bp was obtained by PCR, Which was linkedwith pUM-T.After identified by PCR and digested by enzyme. The sequencingindicted the target DNA was homogous to X14711 by 98.29% .Bovineβ-CN gene 5'regulatory regiongene was successful cloned.3.Construction of recombinant plasmid pcDNA3.1(+)-bβCN-hIL-10: DigestpUM-hIL-10 vector with BamH I and Not I , recover the small fragment, and constructexpression vector pcDNA3.1 (+)-hIL-10 by clone the small fragment into the plasmidpcDNA3.1(+), which was digested by the same enzyme treatment .Then constructepcDNA3.1 (+)-bβCN-hIL-10 plasmid by digest pUM-bβ-CN with Kpn I and BamH I,recovery of large fragments, cloned into the plasmid pcDNA3.1(+)-hIL-10 by the sameenzyme treatment. Identified by PCR and restriction enzyme digestion, the resultsshow that, hIL-10, bβCN gene had been correctly inserted into pcDNA3.1 (+) .4. Microinjection and embryo transfer: 13 superovulation mouse were selected,6mice were seen vaginal suppository, acquired 170 healthy fertilized eggs which wereinjected linearized and purified pcDNA3.1 (+)-bβCN-hIL-10 fragment by microinjec-tion,113 embryos were still healthy after culture in vitro, 113 embryos grow normallyto the morula or blastocyst, developmental rate was 66.5%, that the test eukaryoticexpression vector has little effect on embryonic development, with a certain biologicalsecurity. |
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