Cloning And Characterization Of Fish's Serval Genes Associated With The Immune Response

A cDNA library of grass carp (ctenopharyngodon idellus) leukocytes stimulated with virus was constructed in order to clone and analyze the immune genes which are induced and expressed by virus infection in the immune system of this fish. Initial studies on rapid identification of fish's new genes by expressed sequence tag analysis were also undertook in this paper. Our efforts were taken to lay the foundation of further studies on cloning and function of new genes in fish .The construction and evalution of the A,gtlO cDNA library of grass carp leukocytes are described. Total RNA was extracted from head-kidney leukocytes using Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Poly(A) mRNA was isolated with paramagentic particles method. The cDNAs were synthysized after a series of enzyme-catalyzed reaction and then cloned into XgtlO vector after removing short segments by cDNA sized fractionation columns. The efficiency of packaging is confirmed to be about 4.2 X 107pfu/ug after infecting the host C600hfl. The length of inserted cDNA ranges from 0.4kb to 3kb. It demostrated that the library is of high quality and can be used as a valuable resource for studying gene expression and regulatory mechanisms. Meanwhile, the target cDNA genes such as grass carp interferon (IFN) and Natural killer cell enchacenment factor (NKEF) was probed with PCR amplification.Through intergrated analysis of publice database such as EST, TIGR Gene Index in silico, using human inmune genes sequences that are likely to be present in fish as query, two new fish's genes assosiated with immune response were identified, which including Wilms' tumor related gene (QM) and NKEF. Their full-length cDNA sequences were assembled with fish's ESTs from public EST databases, and then analyzed by softwares such as ORF Finder, Clustal W and GeneDoc.According to the conserved domains of aligned NKEF gene and hum NKEF gene, a pair of primer was designed: NKEF specific forward (5'CATTGGATT TCACCTTTG3'), NKEF specific reverse (5TTCTCCGTGTTTGTCAGT31). Forty cycles of amplification (94 G for 30 seconds, 55 癈 for 30 seconds and 7213 for 1 minutes) were performed in 100 ul reaction mixture (200 mM dNTPs, 1.5 mMMgC12, 10 ul lOxbuffer, 0.2 mM of each primer and 1 unit of Taq polymerase). For further purification. 100 ul of the PCR product was separated using agarose gel electrophoresis. One desired band (about 300 bp) were obtained.EST analysis has been having tremendous impact on the number of biodefense-related genes known in fish. Whilst this will increase over the in coming years, more model organisms are to be fully sequenced and should reveal more genes available to teleost fish. Clearly the coming years will be a major time of gene discovery in fish and most of other organisms, combining lab-working and bioinformatics, and the immunological genetics of fish will benefits from them greatly and make a rapid progress.