| Interleukin-4(IL-4) is a versatile cytokine that play an important role in the regulation of immune responses.1.Rabbit interleukin-4 cDNA from lymphocyte was synthesized by reverse transcription polymerrase chain reaction(RT-PCR) from mRNA extracted from blood lymphocytes of Angora rabbit,which were stimulated by 10 ug/ml Con A in vitro.The rabbit interleukin-4 cDNA from lymphocyte was then subcloned into PMD18-T vector. The sequence of cloned rabbit intreleukin-4 cDNA was determined.The length of the interleukin-4 gene was 444 bp,encoding a peptide of 147 amino acids.the signal peptide contained 24 amino acids and the mature interleukin-4 gene was composed of 123 amino acids.Compared with other mammals in Genbank,including dog,cat,human,pig,cow, horse,goat,panda,rat and so on,the homology of nucleotide sequence was between 57.0 %(panda) and 69.8%(human),the homology of amino acid sequence was between 43.0% (rat) and 53.6%(human).Phylogenetic analysis based on the nucleotide sequence from the interleukin-4 gene showed that Rabbit has the most closed relationgship genetic distance with human.2.By the technology of DNA recombination,the Interleukin-4 cDNA was cloned into eukaryotic expression plasmids pCDNA3.1(+) vector.The eukaryotic expression plasimd pCDNA3.1(+)/IL-4 was successfully constructed.3.The ability of to express full-length rabbit interleukin-4 gene in vitro was assured by RT-PCR in COS 7 cells that were transiently transfected with Interleukin-4 gene entrapped Lipofectamine.The expression products also to increase the proliferation of lymphoblast from the blood of rabbit,which has some biological activities.The Angora rabbit Interleukin-4 gene was cloned firstly in our country,and was eukaryotic expressed in this study.It would provide some evidence for the evolutional relationship of different species,Meanwhile,it provided a basis for further research on immunological and molecular biologic activity and its amplification of recombinant Interleukin-4 protein.
|
PDF Full Text Request