Cloning, Prokaryotic Expression And Tissue Analysis Of Hainan Xinglong Buffalo Disease-related Genes

Xing long buffalo is a native buffalo breed of Hainan province, which has the characteristics of disease-resistant, economic-value high, and muggy-resistant. But the resources of Xinglong buffalo degenerated seriously for the lack of conservation of the breed resources. The degeneration of the breed influenced the buffalo’s performance of production and quality of related product, which brought tremendous economic losses. The cultivation of Xinglong buffalo was threaten by the contagious diseases and reasons above, while the researches of pathological protein of buffalo will help to build up buffaloes’resistance to diseases and direct the future of cultivation.Toll-like receptors (TLRs) are important Pattern recognition receptors (PRRs) in the innate immune system, which can recognize diversified Pathogen-associated molecular patterns (PAMPs) and have been shown to be involved in the damaging inflammatory processes associated with stroke, infection, neoplasia, and other diseases in the central nervous system. Toll-like receptor2(TLR2) is an important member of TLRs family and can control and adjust the innate and acquired immunity, which has the capability to recognize the widest range of PAMPs. TLR2is important for the recognition of mycobacteria and gram-positive bacteria, and it is an important crossroad between encounter with bacteria and development of self-reactive diseases. Myeloid differentiation primary response protein88(MyD88) is a crucial downstream adaptor protein for TLRs, which is essential for TLRs signaling pathway and pro-inflammatory gene expression. The recent studies have indicated that Myd88may play an important role in inflammation and lesion development after traumatic brain injury. For these reasons, the researches of MyD88and TLRs with the pathophysiologic mechanisms that contribute to organ dysfunction will direct therapeutic approaches to ameliorating such TLR-mediated responses that may potentially be of clinical benefit in critically ill patients and livestock. The cDNA of TLR2and MyD88were cloned with standard PCR method from Xinglong buffalo, which has the open reading frames (ORFs) with2355bp and891bp respectively. After the analysis of bioinformatics, the cDNA of MyDSS was constructed into vectors to give a recombinant expression plasmid. Recombinant pET28a-MyD88fusion proteins with His-tag were expressed as Inclusion body in E.coli BL21(DE3) after induction. We purified the recombinant proteins with the Ni-NTA affinity chromatography, and verified the protein by Western blotting with anti-his antibody.The cell divisions cycle42(Cdc42), a member of the Rho family of small guanosine triphosphatase (GTPase) proteins, which plays important roles during cell transformation, proliferation, survival, invasion and metastasis of human cancer cells as a molecular switch. The dysfunction of Cdc42is a feature of some diseases including cancer. Recent studies of Cdc42protein have investigated its roles in pathophysiologic mechanisms and assessed its therapeutic potential in related diseases. The cDNA of Cdc42was amplified with576bp by the method above, the transcription level of Cdc42in heart, liver, spleen, lung, kidney, and intestine were then analyzed with western blotting and real time fluorescence quantitative PCR. Cdc42was found to be expressed in all the tissues above, but at different levels. The expression levels of this gene are highest in lung. The results obtained in this paper provided the base of pathophysiologic mechanisms of related diseases for Xinglong buffalo and direct therapeutic approaches in future.