| Objects Yak(Bos gruniens)is a unique species on plateau,and it can adapt well to the special environments of plateau as a result of its own anatomical and physiological characteristics.Survivin,as the smallest member of the apoptosis inhibitory protein(IAP)family,can participate in both proliferation and apoptosis of cells.Caspase-3,as a key factor in the apoptotic signaling pathway,plays an important regulatory role in disease and apoptosis.This experiment studied the bioinformatics characteristics,the expression variation of survivin in different organs of healthy yaks(newborn group and adult group),analyzed the expression level and unique location of survivin and caspase-3 in immune organs of healthy yaks(newborn group,juvenile group and adult group).These data will provide an important theoretical basis for further studies on survivin regulation mechanism in different organs and tissues,especially in immune organs and tissues of yaks.It provided a sound experimental basis for producing yak antibodies and future research on yak survivin by constructing the survivin recombinant plasmid,inducing expression of recombinant proteins,and preparing polyclonal antibody.Materials and Methods 1.Materials:Different organs and tissues(heart,liver,lung,kidney,brain,testis spleen,thymus and lymph node)of healthy yaks(newborn group,2 – 5 days,n = 5;juvenile group,5-7 months,n = 5;adult group,3-4 years,n = 5);white rabbits,n = 4.2.Methods:(1)RT-PCR was used to clone the coding region of yak survivin gene.Bioinformatics software and official website were used to analyze the target gene.(2)qRT-PCR,Western blot and immunohistochemistry(IHC)technique were used to analyze the expression level of gene,protein and the positive location of survivin in different organs and tissues(heart,liver,spleen,lung,kidney,brain and testis)of yaks in newborn and adult age groups.SPSS21.0 software(one-way ANOVA)was used to determine the significant differences in expression levels of gene/protein of survivin.(3)qRT-PCR,Western blot and immunohistochemistry(IHC)technique were used to analyze the expression level of gene,protein and the positive location of survivin and caspase-3 in immune organs and tissues(spleen,thymus and lymph node)of yaks in newborn,juvenile and adult age groups.SPSS21.0 software(one-way ANOVA)was used to determine significant differences of the expression level of gene and protein of survivin and caspase-3.(4)E.coli expression system was used to induce cloned recombinant plasmid to express the protein.The protein was identified by SDS-PAGE and Western blot.Purified recombinant protein was used to immunize healthy adult white rabbits to prepare polyclonal antibody.Western blot,indirect ELISA and IHC techniques were used to identify the the polyclonal antibodies.Results 1.The coding region of yak survivin gene was successfully cloned(KX121425.1).The protein encoded by the target gene contained 103 amino acids,with a molecular weight 11.8374 kDa.Theoretical isoelectric point was 5.325 pI.12 potential phosphorylation sites were identified.It was a soluble,non-transmembrane protein.The secondary and tertiary structure was predicted based on the gene sequence.From the above findings,survivin was only highly conserved but also exhibited specie specificity in the process of biological evolution.2.Survivin was expressed in all organs and tissues of yak in two age groups.Within the same organ,the expression level of survivin was higher in the adult group than that in the newborn group.Within the same age group,survivin was consistently expressed at relatively high levels in the heart and liver.Immunohistochemistry results showed that survivin positive localization was mainly concentrated in the cytoplasm.Within the newborn group,survivin was mainly located on the cardiomyocytes of the heart,the hepatocyte of the liver,the red pulp of the spleen,the bronchi epithelial cells of the lungs,the proximal and distal tubules of the kidney,the nerve fibers of the brain,and the myoid cells of the testes.The localization of survivin in adult group was somewhat different from newborn group: in the adult yak lung,survivin was detected not only in the cytoplasm of all levels bronchi epithelial cells,but also in the alveolar epithelial cells;in the adult yak testes,survivin was mainly expressed in the spermatogenic cells.3.The expression of survivin in immune organs of yaks(newborn group,juvenile group,and adult group)showed that in the same age groups,the expression in the spleen was the highest,followed by thymus,and lymph node.The expression of survivin mRNA in the spleen and protein in the lymph node was significantly higher than that in the others(P < 0.01).Within the same immune organs,the expression of survivin decreased rapidly with age(P < 0.01).The expression of caspase-3 was roughly opposite to that of survivin.Immunohistochemical results showed that the positive location of survivin and caspase-3 was same.They were mainly distributed in the medulla of lymph nodes,the thymus bodies of the thymus,the red pulp and marginal regions of the spleen.4.The recombinant plasmid was successfully constructed.The target protein was an about 16.5 kDa inclusion bodies,as confirmed by SDS-PAGE and Western blot after IPTG induction.Western blot results showed that the purified protein could be combined with positive serum,indicating its good reactogenicity.Again,it showed positive results when the serum obtained by immunizing rabbits with recombinant protein can react with the recombinant protein,and this also indicated its good immunogenicity.Besides,it proved that the prepared polyclonal antibody had good antibody titer by indirect ELISA.The prepared polyclonal antibody can be used in immunohistochemical Conclusion 1.The result which was analyzied by the bioinformatics characteristic method showed that survivin was not only highly conserved but also exhibited specie specificity in the process of biological evolution. 2.The expression and localization of survivin in different organs of newborn and adult yak were analyzied to speculate that survivin may participate in functional adjustment in different organs and tissues.These data will provide an important theoretical basis for further studies on survivin regulation mechanism in different organs and tissues of yaks. 3.The expression and localization of survivin and caspase-3 in immune organs of yak were analyzied to speculate that survivin may participate in immune organs' adjustment in caspase-3 mediated apoptosis pathway and delay immune organs' aging,which provided an important theoretical basis for further study on survivin mechanism in regulation of yak immune function. 3.The recombinant plasmid(yak survivin)was constructed,induced to express the recombinant protein,and used to produce rabbit polyclonal antibody.The antibody can be used in immunohistochemistry,Western blot and indirect ELISA.This experimental provide a sound experimental basis for producing yak antibodies,and it also provided a theoretical basis for the future research on yak survivin. |
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