Study On Function Of Recombinant Bpi Protein Of Bashibai Sheep And Its Hybrid Sheep

Bactericidal/permeability increased protein(BPI) is a kind of cationic antimicrobial proteins and was found in the polymorphonuclear granulocyte of human and mammal, BPI protein can specific killing of gram-negative bacteria and neutralize endotoxin, it also has the biological founction of immune regulation, induced apoptosis of endothelial cells, antifungal, anti-toxoplasma infection and so on.Objective:The article focuses on Bashiby and hybrid sheep(argali♂× Bashiby ♀) applying molecular biotechnology to clone and express the BPI gene of Bashiby and hybrid sheep in P.pastoris, studying the inhibition of recombinant BPI on gram-negative bacteria, the influence on apoptosis of endothelial cells, and the effection on MO(Mycoplasma ovipneumoniae, MO) in vitro,and compare the differences of BPIm RNA expression level between Bashiby and hybrid sheep,so as to reveal the biological function of BPI protein.Methods:(1)Cloning and eukaryotic expression of the BPI gene in Bashiby and Hybrid sheep:Total RNA was extracted from Bashiby and hybrid sheep polymorphonuclear neutrophils(PMN)and was amplified to BPI gene fragment by RT-PCR.Then the BPI gene fragment was cloned into p PIC9 K vector to construct a eukaryotic expressing plasmid p PIC9K-BPI. After identified by PCR,restriction analysis and sequencing,the constructed recombinant plasmid p PIC9K-BPI was transformed to P.pastoris GS115 by electrotransformation,and the expression was induced by methanol.Finally, using the SDS-PAGE to identify the expressed product.(2) Purification of the expressed product of BPI gene in Bashiby and its Hybrid sheep: In order to obtain the higher purification of recombinant bectericidal/permeability increasing protein(r BPI) of Bashiby and Hybrid sheep,using the Ni2+ chelated affinity chromatography to purify r BPI and identified by SDS-PAGE.(3)Detecting the inhibition of r BPI of Bashiby and Hybrid sheep:The purification of r BPI added into E.coli,Salmonella and Staphylococcus, using the broth microdilution method to detect the inhibition of three different pathogenic bacteria.(4)Detecting the apopotosis of MBECs(mouse brain endothelial cell)induced by Bashiby and Hybrid sheep r BPI:Adding r BPI of Bashiby and Hybrid sheep to MBECs(mouse brain endothelial cells) cultured in vitro, the method of Annexin V FITC/PI staining were used to identify apoptotic cells under the fluorescence microscope and the apoptosis rate was investigated with cell counting, then DNA ladder were detected by agarose gel electrophoresis.(5)The effects of r BPI of Bashiby and Hybrid sheep on MO cultured in vitro:Adding r BPI to MO and detected the expression level of 16 S r RNA by real-time PCR.(6)Detected BPI m RNA expression level of neutrophils in Bashiby and its Hybrid sheep: Respectively adding MO to the Bashiby sheep neutrophils which were isolated cultured in vitro then detected BPI m RNA expression level of neutrophils by real-time PCR.Results and conclusions:(1)Successfully constructed BPI eukaryotic expression vector of the Bashiby sheep and Hybrid sheep, successfully expressed in Pichia pastoris and obtain 43 KD r BPI of Bashiby sheep and Hybrid sheep.(2)The purified products were analysed by SDS-PAGE, the strip single target band could be observed, the recombinant BPI protein of Bashiby sheep and Hybrid sheep were successfully purified.(3)Both Bashiby and Hybrid sheep’r BPI could inhibit the gram-negative bacteria,but had on effect on gram-positive bacteria. More than 20μg/ml of Bashiby sheep r BPI can significantly inhibit the growth of Ecoli and Salmonella than the control group, and more than40μg/ml of Hybrid sheep r BPI can significantly inhibit growth of Ecoli and Salmonella than the control group.The results showed Bashiby sheep r BPI could better inhibit the growth of gram-negative bacteria than Hybrid sheep r BPI.(4) While observed by the method of Annexin V FITC/PI staining,added Bashiby sheep r BPI concentration of 180,250,540nmol/L, the apoptosis rate(14.68%,20.46%,30.42%) were very significant higher than the control group(p<0.01,p<0.01,p<0.01), and DNA ladders of MBECs were clearly detectable treated at 540nmol/L of Bashiby sheep r BPI;when added Hybrid sheep r BPI concentration of 250,540nmol/L,the apoptosis rate(14.76%,11.03%) were significant higher than the control group(p < 0.05,p < 0.05), and not DNA ladders of MBECs was clearly detected by agarose gel electrophoresis.(5)The 16 s r RNA expression level of MO which had been treated by Bashiby sheep r BPI at 4h was significantly lower than the control group( p<0.05); while treated by Hybrid sheep r BPI, the 16 s r RNA expression level of MO had no significant with control group. It showed that Bashiby sheep r BPI could inhibit the growth of MO, while Hybrid sheep r BPI had no effect on MO.(6)After infected by MO from 4h to 8h, the BPI m RNA expression level of neutrophils of Bashiby sheep treatment group were higher than Bashiby sheep control group and Hybrid sheep treatment group( p<0.05, p<0.05), it showed that the BPI m RNA expression level of neutrophils in Bashiby sheep could significant improved by the infection of MO, while the BPI m RNA expression level of neutrophils in Hybrid sheep had no significantly improved.