Polymorphism Analysis Of The ISG15 Gene And Inducible Expression In The Hu Sheep

Because the ISG15 gene plays an enssencial role in broad-spectrum anti-viral activities, against the HIV, A/rWSN influenza virus, B/Lee influenza virus, HSV-1, HBV, JEV, RSV, VAVA, recombinant Sindbis virus, hepatitis C virus, Ebola VP40 virus, bovine viral; anti-bacterial infection and acquired immunity, the ISG15 gene was selected as a candidate gene in the Hu sheep in the current project. According to bioinformatics method, the ISG15 gene in the Hu sheep was cloned to get the DNA sequence. By PCR-SSCP analysis, ISG15 inducible expression was developed and the relationship between single nucleotide polymorphism and immune traits in Hu sheep were investigated to screen out the molecular marker associated with immune traits for sheep breeding.1. Cloning and Bioinformatics analysis of ISG15 genes in Hu sheepBasing on the GenBank (FJ844480.1) standard DNA sequence of login sheepe, one pairs of primers of ISG15 gene were designed to clone ISG15 gene of Hu sheep. The 1SG15 gene of Hu sheep was cloned,2511 bp DNA in length, including of 5'-UTR(1444bp), ORP1(3bp), Intron(446bp), ORF2(471bp) and 3'-UTR(68bp),and which codes 157 amino acids. The molecular weight of ISG15 proteins in Hu sheep was 17.4 KDa and its isoelectric point was 6.29. The ISG15 protein structure is four N-glycosylation sites, six phosphorylation sites. Hu sheep ISG15 protein presumed existence of multiple functional sites including 14-3-3₃ signaling molecules or functional motifs including GSK3 phosphorylation site recognition.2.ISG15 PCR-SSCP detection of single nucleotide polymorphism and analysis of population genetics in the Hu sheepIn this study, SNPs of the whole cloned ISG15 gene was detected in Hu sheep by PCR-SSCP method. Experimental design of 10 pairs of primers detected 15 mutated sites in addition to U4, U6, U7 primers. There were 152,155,158,239,444,713,726,797,818, 862,1077,1772,1909,2171,2305 and 2405 sites and showed 3 genotypes.152,155,239, 444,713,726,862,1772 and 1909 sites were mained in wild-type alleles, and 158,797, 818,1077,2171,2305 and 2405 sites dominated the mutant allele. Most ISG15 SNP PIC are moderate polymorphism beween 0.25 and 0.38.χ2 test, results showed that the 10 of 16 mutation bits were in the Hardy-Weinber balance, but 155,239,797,818 and 1077 bit in the UTR and 2405 bit in the ORF2 were not in Hardy-Weinberg balance state, these sites which may be natural selection, random drift cause the mutation rate increase. There results expected to get more choice effects.3. The ISG15 gene inducible expression in the Hu sheepSelected 10 Hu sheep fibroblast cell culture, poly I:C induced cells were cultured for 3,12 and 24 h, extraction of RNA,β-actin, IFNA1 and ISG15 were examined by using real-time quantitative PCR. Theβ-actin and ISG15 expression was much higher compared with IFNA1 expression,β-actin and ISG15 Ct value appeared in between 18 to 33 cycles, and the vast majority Ct value of IFNA1 appeared in 39 cycles later, the test could only analyze ISG15 relative toβ-actin expression., ISG15 relative to the housekeeping geneβ-actin expression level began to rise at the first 3 h by Poly I:C induction; the ISG15 relative to theβ-actin expression level has reached nearly 10 times at the 12 h; and ISG15 relative toβ-actin expression increased slightly and reached the maximum at the first 24 h.However, the expression peak of the ISG15 gene relative to the basal levels appeared in the first 12 h and an expressional variation at this time point existed in terms of different time for each individual. It suggested that the time expression was not consistent individually.