Expression,Purification And Preliminary Studies Of Hgp44 Hemagglutination Domain From P.Gingivalis And The Study Of Adipose Tissue Targeting Ability Of Salmonella VNP20009

Porphyromonas gingivalis,an anaerobic gram-negative bacterium associated with chronic periodontitis,can agglutinate human erythrocytes.In general,hemagglutination can be considered the ability to adhere to host cells;however,P.gingivalis-mediated hemagglutination has special significance because heme markedly accel-erates growth of this bacterium.A number of studies have indicated that a major hemagglutinin of P.gingivalis is intragenically encoded by rgpA,kgp,and hagA。rgpA encoded arginine-specific gingipains(Arg-gingipain A,RgpA)and kgp encoded lysine-specific gingipain(Lys-gingipain,Kgp),both gingipains are secreted protease,are Porphyromonas gingivalis’s major virulence factors.The genes rgpA and kgp have similar structures,with the encoded proteins consisting of an amino-terminal propeptide region,a proteolytic domain and carboxy-terminal adhesin domains[Hgpl5(HbR),Hgp17,Hgp27 and Hgp44].Adhesin domains are also encoded by the hemagglutinin A-encoding gene hagA;These regions are quite similar to the carboxy-terminal regions(Hgp44 and Hgp15)of rgpA and kgp.Further studies showed that Hgp44 is the main hemagglutination domain.In this study,we cloned the truncated Hgp44 gene,and fused with ELPs.The tHgp44-ELPs was expressed in E.coli and purified by inverse transition cycling.We demonstrated in this study that recombinant tHpg44-ELPs had hemagglutinating activity.In the second part,we studied the adipose tissue targeting ability of Salmonella VNP20009.Attenuated Salmonella Typhimurium VNP20009 can preferentially accumulate and replicate in hypoxic tumor region and suppress the growth of the tumor,is a typical representative of tumor-therapeutic bacteria which is rapidly developed recently.And it is now widely used as an anticancer agent.Many studies also show that adipose tissue of obese mice is hypoxic.Tissue hypoxia was confirmed by an exogenous marker,pimonidazole,and by an elevated concentration of lactate,an endogenous marker.This is similar to the hypoxia area of tumor tissue.Our study shows that VNP20009 have the ability targeting to the adipose tissue of obese mice,and not limited to specific obesity model.However,the bacterial titer of adipose tissue was lower than tumor tissue.Further studies showed that the fat homogenate can slow down the growth rate of Salmonella.This shows that the process of VNP20009 targeting adipose tissue is affected by the positive and negative two forces.Hypoxia is a positive factor and fat is a negative factor.This suggests that,if we want to further improve the ability of the VNP20009 target to adipose tissue,it is necessary to carry out certain genetic transformation.