| Mycoplasma bovis (Mb) is one of an most important and emerging pathogen which can induce pneumonia, arthritis, mastitis, rabortions and infertility in cattle.lt is the first time that Mb was isolated from the lungs of calves suffering from pneumonia in domestic in2008, then began to spread in our country, the huge economic losses has been caused by Mb at home and abroad at present. On the basis of preliminary studies, This article analyze the protein composition of whole cell proteins and membrane proteins and western blot in order to further filter the advantages of epidemic isolates and found the immunogenicity fragment of the antigen protein.lt can provide a theoretical basis for the ELISA diagnostic kits and vaccine development,and also can provide a theoretical basis for the further prevention and control of Mb and orther mycoplasma disease.1The study on expansion of cultivation, concentrations determination and PCR identification of MbThis test was used the improvement mycoplasma medium for expand cultivation of six Mb isolates which isolated from all over the country, PCR identification by specific primers which based on conserved genes of Mb,and concentrations of pure cultures were measured by ColorChange Units(CCU). The reslut showed that the color of liquid medium began to change in24h and reached the logarithmic growth phase in48h, the cultures of six isolates were amplified the fragment as expected size (1912bp),the concentrations of the pure cultures of six isolates were all above108CCU/ml. The study found that the improved liquid medium was suitable for the growth of Mb and shorter culture time was consumed, the PCR identification of Mb established in this study were good specificity, high sensitivity and simple operation,to prepare for later study on whole cell protein and membrane protein.2The analysis of whole cell protein of different Mb isolatesAnalysis and comparison the different of protein peptide between different isolates of Mb and combined with preliminary-test results of growth characteristics and pathogenicity to further screening of the advantages of epidemic isolates. In this study,we extracted the whole cell proteins of six Mb isolates(W70isolate,1738isolate,Q3isolate,Ql isolate,JX02isolate,677isolate) which isolated from all over the country by using the method of lysates.lt was showed that there was differences on protein polypeptide number and protein polypeptide definition of six Mb isolated by12%SDS-PAGE and coomassie brilliant blue staining,and there were the same protein polypeptide number and protein molecular weight within W70isolate,1738isolate,Q3isolate,Ql isolate,the total protein polypeptide number was17and the range of molecular weight was among130.8kDa-23.2kDa; Compared with the first four isolates,the same of protein polypeptide number and protein molecular of JX02isolate,but lower expression protein bands; protein polypeptide number and the expression of protein bands of677isolate was significantly lower than the previous four isolates. The study showed that the protein expression of W70isolate,1738isolate, Q3isolate and Ql isolate was better than JX02isolate and677isolate,it can be used for candidate isolate of future study.3Comparison of six methods of membrane protein extraction on Mb isolatesIn order to comparied different methods of membrane protein extraction and filter out the bett er membrane protein extraction methods and extraction parameters,this study using six methods of membrane protein extraction extracted membrane protein of three Mb isolate (W70isolate,1738isolate, Q3isolate), the methods indued different concentrations of TritonX-100(0.2%、0.4%、0.6%、0.8%), different concentrations of TritonX-114(1%、2%、3%、4%),different concentrations of TritonX-100combined with different concentrations of KI (0.6M,0.8M,1.8M), different concentrations of TritonX-114combined with different concentrations of KI (0.6M,0.8M,1.8M), repeated freezing and thawing and ultrasound, membrane protein obtained by each method analyzed by12%SDS-PAGE electrophoresis and Bradford quantitative showed that:(1) there was no significant difference in SDS-PAGE of membrane proteins and protein bands were basically the same between3Mb isolates, but there were significant differences in the number of protein bands of membrane proteins and protein concentration on the same isolate of different membrane protein extraction (P<0.05).(2) At the methold which combined with KI, there were significant difference in the impact of protein concentration extrated by different concentrations of KI (P＜O.01) membrane proteins extrated by0.6M KI was better then0.8M KI on the number of protein bands and protein concentration,while0.8M KI was better then1.8M KI which was the worst.(3) Comprehensive show that membrane proteins extrated by3%tritonX-114and0.4%Triton X-100combined with0.6M KI are better extraction of membrane proteins, the protein bands were as high as26-28and24-28respectively,and protein molecular were among167-11.4kDa and 170kDa-12.2kDarespectively,the major protein were101.4kDa、92.9kDa、85.CkDa、77.9kDa、71.3kDa、65.3kDa、45.9kDa、38.5kDa、33.8kDa、29.6kDa、27.1kDa、22.7kDa、20.8kDaand101.4kDa、92.9kDa、77.9kDa、74.5kDa、65.3kDa、59.8kDa、52.4kDa、38.5kDa、33.8kDa、29.6kDa、24.8kDa、21.6kDa、19.9kDa respectively.(4) The effect of membrane proteins extracted by repeated freezing and thawing as well as ultrasound was not good. Studies have shown that3%tritonX-114and0.4%Triton X-100combined with0.6M KI were the optimum membrane proteins extraction method of Mb, this study complements the part of the contents of the membrane protein extraction of Mb in domestic and to enrich a part of the parameters of membrane protein extraction of Mb at abroad.4Western blot analysis for whole cell protein and membrane protein of Mb isolatesIn order to find out the protein antigen fragments of whole cell protein and membrane protein of Mb,in order to provide a theoretical basis for the ELISA diagnostic kits and vaccine development,this study using mouse antiseum for western blot that analyzed whole cell protein of six Mb isolates (W70isolate,1738isolate,Q3isolate,Ql isolate,JX02isolate,677isolate) as well as membrane protein of three Mb isolates (W70isolate,1738isolate,Q3isolate) extracted by3%tritonX-114,0.4%tritonX-100,3%tritonX-114or0.4%TritonX-100combined with0.6M Kl,repeated freezing and thawing and ultrasound.The result of western blot showed that:(1) Whole cell protein of six Mb isolate had two common hybridization band which molecular is55kDa and43kDa respectively (the range of erro is±lkDa the same below), Expression of hybridization band of JX02isolate and677isolate was weaker than other four isolates.(2) there have three common hybridization band on membrane protein of three Mb isolates (W70isolate,1738isolate,Q3isolate) extracted by3%tritonX-114and0.4%TritonX-100combined with0.6M KI, molecular of hybridization band were55kDa,43kDa and18kDa respectively.(3) there have two common hybridization band on membrane protein of three Mb isolates extracted by o.4%tritonX-100and3%TritonX-114combined with0.6M KI as well as repeated freezing and thawing and ultrasound, molecular of hybridization band were55kDa and43kDa respectively. Studies have shown that:(1) There were significant hybridization band appeared when whole cell protein and membrane protein of Mb isolates reacted with antiserum.and with good immunogenicity; the number of hybridization band of membrane protein extracted by different extraction mehods was different.(2)whole cell protein of six Mb isolates and membrane protein of three Mb isolates of different extraction mehods have the same westblot hybridization band(55kDa and43kDa),it was showed that55kDa and43kDa were the major antigenic protein of Mb.This result provide a theoretical basis for vaccine development in coming future. |
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