Preparation Of Recombinant MBL And Response Analysis Of Resistance To Sheep Disease

Mannan-binding lectin (mannan-binding lectin, MBL), also known as mannose-binding lectin(mannose-binding lectin, MBL), plays an important role in the natural immune system of the organism. Bythe way of complement activation without relying on antibodies, the protein can start complement systemand play a part in the immune conditioning, hence，it is critical of resisting pathogens in infected areas.Study find that MBL function is dependent on integrated structure. MBL gene mutation leads to significantreduction of MBL level in serum,the individuals which have low levels of MBL are suceptible topathogens. Futhermore, sheep mycoplasma pneumonia has caused serius injury to sheep raising ofXinJiang. In this study, we took MBL、sheep and Mycoplasma Ovipneumoniae（MO) for the object toexplore MBL resistance to Mycoplasma pneumonia and other relevant diseases. This research provides atheoretical basis for the development and research of drug treating Mycoplasma pneumonia. The resultsas follows:(1)The changes of immune response of Chinese Merino sheep which had been infected MO were detectedwith ELISA: we selected16healthy Chinese Merino sheep as subjects,3of which were the controlgroup.The13sheep as experimental group were artificially infected Mycoplasma ovipneumoniae,beforechallenge1d, after challenge1d,7d,14d,21d,serums were gathered to detect the level of immune factorsin different individuals by ELISA, such as TNF-α, IFN-γ, complement C1, C3.We conclude by results thatthe extent of Mycoplasma pneumonia could be infered through the changes of the above factors(2)Change of MBL in serum and histopathologic analysis after artificially infecting Mycoplasmaovipneumoniae: infected sheep were slaughtered to study the relationship of MBL level and Mycoplasmapneumoniae by histopathological analysis, the results showed the higher levels of serum MBL beforechallenge histopathological, the lower scores, that is to say, MBL has certain resistance to Mycoplasmapneumoniae.(3) The preparation of natural and recombinant MBL: PEG-6000was used to extract natural MBL fromsheep serum. pPIC9K eukaryotic expression vector and cloning vector pMD18-T-MBL were digested byNotⅠ, SnaBⅠ, respectively. Then, the recombinant expression vector pPIC9K-MBL was got by T4DNAligase, which was transformed into Pichia pastoris GS115, natural MBL and recombinant MBL wereidentified by SDS-PAGE. The results are that natural MBL and recombinant MBL appeared in predictedbands. Conclusions: MBL which derived from sheep serum was impure; recombinant MBL that was gainedby pPIC9K-MBL was little and insufficient for subsequent purification.(4) At the cell and individual levels, the effects which had been had on immune factors, including TNF-α,IL-2, by human MBL and sheep MBL were studied after infecting Mycoplasma pneumoniae: Beforeinfecting, human MBL and sheep MBL were injected to experiment groups, separately. The results showed,At the cell level, the difference of TNF-α variation was significant among human MBL、sheep MBL andexperimental control group at24h, the difference between experimental control group and experimentalgroup was also significant at48h and72h. and the difference of IL-2variation was significant amonghuman MBL、sheep MBL and experimental control group at24h、48h、72h. At the individual level, thedifference of TNF-α variation was significant from1d to21d among the three groups, and the difference ofIL-2variation was significant at7d among the three groups, the difference between experimental controlgroup and experimental group was also significant at14d, but the difference in experiment was notsignificant, the one among the three groups was not significant either. We reached the decision that MBLcould enhance the anti-inflammatory ability of the organism.