Cloning And Sequencing Of Grass Carp Interleukin-2 Gene And Its Expression In Prokaryocyte

Interleukin-2(IL-2) is a lymphokine which plays pivotal role in the regulation of intricate immunology network. It has been thoroughly studied and wildly aplicated in human and other vertebrates, but the studies on lower vertebrates such as fish are still very poor. In the progress of evalution, fish connects the vertebrates with invertebrates, so it is of interest to investigate the IL-2 in fish which will be helpful to understand the origin and phylogency of IL-2.In this research, a grass carp (ctenopharyngodon idellus) IL-2 was illustrated. First of all, concanavalinA (ConA) was used to stimulate the grass carp pronephric leucocyte in vitro. In the supernatants of cell cultures, activaties of promoting the proliferation of mitogen stimulated grass carp lymphoblasts have been found. Evidence for the existence of grass carp IL-2 was primarily observed.To go deep into the grass carp IL-2, a cDNA coding grass carp IL-2 was cloned and sequenced. After 14 hours' inductions of phytohaemagg-lutinin (PHA) and ConA cooperated with PMA, the total RNA was extracted from grass carp proniphric leucocyte. The mRNA was trasncripted into first strand cDNA by oligodT(ig) primers, which was served as a template for reverse transcription-polymerase chain-reaction (RT-PCR). The RT-PCR primers were designed based on the conservative region of the 5'and 3'-terminal of human and other vertebrates' IL-2 cDNA gene. The amplification conditions were optimized and than specific product was obtained by elevating the annealing temperature to 56癈. After purification, this RT-PCR product which was about 500bp shown in 1.5% agrose gel electrophoresis was inserted into the pUCm-T plasmid by T-A cloning and sequenced for necleotide sequences using T7 promoter primerby ABI377 DNA sequencer. The full length of grass carp IL-2 cDNA gene was 492bp coding for 164 ammo acid protein whose molecular weight was calculated to be about 17.5kD. No cysteine residue but two potential N-glycosylation sites were observed in the deduced polypeptide sequence which suggested that grass carp IL-2 was a glycoprotein without potential S-S structure. Analysed with DNAMAN and OMIGA software, we found that Grass carp IL-2 has an nucleotide sequence homology of 34-40% with human and other vertebrates and an amino acid sequence homology of 23% with ftatfish(Paralichthys sp\Grass carp IL-2 cDNA gene fragment was cut out from pUCm-T plasmid by Ncol and BamHI enzyme and then ligated to pET28b express plasmid. The recombinant plasmid pET28b_IL-2 was selected and transferred to E.coli. BL21/DE3. After the induction of IPTG, a specific protein band with expected molecular weight of 21kD were detected by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) analysis. This result indicates that grass carp IL-2 can be expressed in prokaryocyte which might be a basis for further studies on its immuology function .