Cloning And Expression Of The Hemolysin-Related Protein Hcp In Pseudomonas Plecoglossicida

Pseudosciaena crocea is one of the important commercial fishes for mariculture in China.It has been favored by consumers because of fish with its delicacy and abundant nutrition.However,in recent years,with the continuous expansion of mariculture,the upgrading and development of the industrial structure of P.crocea,the diseases of P.crocea has become the major obstacles development of P.crocea mariculture.P.crocea "white-spots disease" is an important bacterial disease affecting the P.crocea mariculture in recent years.In this study,we were using intraperitoneal injection way artificially infected the P.Crocea to observe the distribution of the pathogen of white-spots disease of P.plecoglossicida in the P.crocea by immunohistochemical and tissue pathological slices Technology;The clone of Pseudomonas plecoglossicida Hcp and its expression;the recombinant protein Hcp in the form of inclusion body is obtained;after protein refolding,the soluble protein be obtained successfully,which can be provided experimental data for further research on the invention and development of the the T6 SS of P.plecoglossicida.Healthy P.aeruginosa is infected by artificially intraperitoneal injection,the result shows P.aeruginosa had typical symptoms of "white-spots disease" : the diseased fish had no itality and was unresponsive.Anatomical findings revealed that the internal organs such as the spleen,kidney,and liver were covered with white spots and accompanied by symptoms of ascites.The diseased fish showed obvious damage to the visceral tissues such as fish liver,spleen and kidney,showing different degrees of hyperemia,hemorrhage,necrosis by observation on histopathology.As the bacteria mass multiplication,necrotics and dissolutions of cells in the body of multiple organs of P.aeruginosa,eventually the diseased fish to die due to the failure of multiple tissues and organs.The results of immunohistochemical staining showed that the strong positive signal of the pathogen was in the liver,spleen and kidneys,particularly in macrophages of the internal organs of diseased fish.Studies shown that P.plecoglossicida could infected multiple tissues and multiple system organ.The transport gene Hcp in T6 SS of P.Plecoglossicida system was cloned and expressed by molecular biology technique.The weight of Hcp,which we obtain by PCR are 490 bp,molecular weight was about 25 kDa by SDS-PAGE.The recombinant protein pET-30a-Hcp is mainly expressed in the form of inclusion bodies.Renaturation and purification of Protein pET-30a-Hcp,the results of Western-blot showed that the recombinant protein pET-30a-Hcp is capable of inducing immunity.This study laid the foundation for further study on the pathogenic mechanism of Pseudomonas sinensis and its translocation gene Hcp in T6 SS.