Screening Of Brucella Strong Promoter And Expression Of Recombinant Foot-and-Mouth Disease Virus VP1 Gene

Brucellosis is an important zoonotic disease caused by brucella.In 2012,the State Council issued the National Medium-Long Term Animal Disease Control Program(2011-2020),The cloth disease as a priority prevention and control of domestic 16 kinds of animal disease,and as the current prevention and control work of the most important.The disease poses a serious threat to animal husbandry and human health,and the use of vaccine immunization is one of the important measures to prevent brucellosis.At present,vaccine for the control of brucellosis is mainly S2(bv.2)in China,which has a weak virulence,good immune effect,high security features.Brucella can cause the highly Th1 immune response,which makes brucella become the ideal heterologous antigen vector,in order to develop a Brucella live vector vaccine with highly efficient expression of foreign gene,in this subject promotor Psod,Pdnaj and Pdnak were cloned into YGTpBBR1mcs2 by using green fluorescent protein as the reporter gene.The recombinant expression vectors YGT-pBBR1mcs2-Pdnaj,YGT-pBBR1mcs2-Psod and YGTpBBR1mcs2-Pdnak were transformed into brucella,and the expression of GFP protein was detected by fluorescence microscopy.The results showed that the promoters of three different intensities could start the expression of GFP gene in brucella S2 and the promoter ability from strong to weak is Pdnak,Pdnaj,and Psod.Foot and mouth disease(FMD)is an acute,heat,and highly contagious infectious disease caused by foot and mouth disease virus(FMDV).This disease can cause blister the cloven-like animals,Foot and other parts of the skin blisters and cause some animal deaths,seriously affecting the development of animal husbandry industry.VP1 protein is the main structural protein of FMDV,which is also the key to virus infected cells,and has the site of binding to cell receptor.Therefore,VP1 gene is the preferred target antigen for gene engineering vaccine.In this study,the brucella S2 strain was used as the parent strain,and the VP1 gene expression cassette of FMDV was used to replace the Brucella wboA gene by using the sucrose suicide plasmid vector.The recombinant foot-and-mouth disease virus VP1 gene was successfully constructed in live vaccine.The VP1 gene of the recombinant strain was detected by fluorescence quantitative PCR.It was found that VP1 gene had significant transcriptionalchanges in the host bacteria,indicating that the VP1 gene of foot and mouth disease virus was expressed at the mRNA level.The results showed that there was about a band of 30 KD in size in the recombinant strain compared with the parent strain,which was consistent with the theoretical size of VP1 protein,and proved that the foot and mouth disease virus VP1 expression in Brucella.In this study,the promoter of Brucella was screened and its strength was verified.The method of expressing exogenous gene with Brucella as a vector was established,and laid the foundation for further brucella vaccine research.