Cloning, Expression And Characteristics Of Argali Hybrid Sheep SPLUNC1 Genes

The Argali(♂) and Bashiby sheep(♀) hybrids have the characteristics of large size, lamb growth fast, Lean higher,but the hybrids lamb susceptible mycoplasma pneumoniae and death. short palate,lung and nasal epithelium clone 1 was a newly discovered bioactive substances with host defense functions.Some scholars have found that SPLUNC1 gene is congenital host defense mycoplasma pneumoniae infection in key genes.Object: This paper is to clone Argali hybrid offspring short palate, lung and nasal epithelium clone 1(SPLUNC1)gene, obtain the c DNA full- length sequence, comparative study between Argali hybrid and Bashibai sheep,and the SPLUNC1 gene eukaryotic expression,study the bacteriostatic action of r SPLUNC1 protein, in vitro cultivation of mycoplasma pneumoniae sheep effects(Mo) growth, the influence of neutrophil elastase activity and lymphocyte apoptosis of respiratory pathogenic bacteria infection, comparison of the functional differences between argal hybrid sheep and bashiby sheep r SPLUNC1 protein, laying foundations for r SPLUNC1 protein function in vitro.Methods:(1)Amplification SPLUNC1 c DNA full- length of Argal hybrid sheep: Using RT-PCR and rapid amplification of c DNA ends to clone 5’end sequence and 3’end sequence of SPLUNC1 of Argal hybrid sheep, sequencing and splice to get full- length sequence.(2) Bioinformatic analysis of Argal hybrid sheep SPLUNC1 c DNA:Using bioinformatics online analytical tool and software to analysis Argal hybrid sheep SPLUNC1 nucleic acid,encoding amino acid,protein signal peptide, subcellular localization,advanced structure and phylogenetic analysis.(3)Argal hybrid sheep r SPLUNC1 eukaryotic expression and purification of protein: According to the SPLUNC1 gene c DN A sequence design primers specific expression, using RT-PCR to amplify the open reading frame sequence of Argal hybrid sheep SPLUNC1 gene, to construct recombinant expression plasmid p PIC9K-SPLUNC1,the recombinant expression plasmid were transformered into Pasteur pichia GS115, and by using the methanol recombinant strain GS115/p PIC9K-SPLUNC1 inducing expression, analyzed by western blot and SDS-PAGE,using N i-NTA to purified the recombinant SPLUNC1.(4)Argal hybrid sheep r SPLUNC1 bacteriostatic action:The purification of Argal hybrid sheep r SPLUNC1 added into E.coli,Streptococcus pneumoniae,pasteurella and staphylococcus aureus,using the broth microdilution method to detect the inhibition of pathogenic bacteria.(5) Argal hybrid sheep r SPLUNC1 effect on sheep mycoplasma pneumoniae growth: Using the methods of plate colony counting and real- time fluorescent quantitative PC R(FQ- PCR) to detect Argali hybrid sheep r SPLUNC1 effect on the in vitro culture of Mo growth.(6) Argal hybrid sheep r SPLUNC1 effects on neutrophil elastase activity: Using the method of four peptide substrate to detect Argali hybrid sheep r SPLUNC1 effects on neutrophil elastase activity.(7) Argal hybrid sheep r SPLUNC1 affect respiratory pathogenic bacteria infection of lymphocyte apoptosis:Using the method of DN A ladder and Annexin V-FITC /PI double staining to detect Argali hybrid sheep r SPLUNC1 effects respiratory pathogenic bacteria infection of lymphocyte apoptosis.Results and conclusion:(1) The c DNA length of Argali hybrid sheep SPLUNC1 gene was 1117 bp.(2) The Argali hybrid sheep SPLUNC1 gene Open Reading Frame was 768 bp, encoding 255 amino acids, predicted molecular weight 26.57 k D and an isoelectric point of 5.006. Bioinformatics analysis showed that SPLUNC1 protein N end have 20 amino acid residues signal peptide, with high hydrophobicity,were located extracellularly.(3) Successfully constructed SPLUNC1 eukaryotic expression vector of the Argali hybrid sheep,and expressed in Pichia pastoris and obtain 25.96 KD r SPLUNC1.(4) Concentration 20μg/m L-40μg/m L of Argal hybrid sheep r SPLUNC1 can extremely significant(p<0.01) inhibit the growth of pasteurella and Ecoli.(5)Concentration 40μg/m L Argal hybrid sheep r SPLUNC1 can significant difference(p<0.05) inhibit the growth of mycoplasma in agar plate.The 16 s r RNA expression level of Mo which had been treated by Argal hybrid sheep r SPLUNC1 at 4h was extremely significant( p<0.01) lower than the control group.(6)The results showed that more than 20μg/m L Argal hybrid sheep r SPLUNC1 can enhance the activity of the nw-trending, with dose effect.(7) The results DNA ladder and Annexin V-FITC/PI double staining showed that Argal hybrid sheep r SPLUNC1 no effect lymphocyte apoptosis with pathogenic bacteria infection.