Cloning, Expressing And Identification Of Aspartic Protease Gene And Cathepsin B Protease Gene From Ancylostoma Caninum In E.coli

Hookworm is a important intestinal parasitic nematodes in human,in order to ensure children grow healthily and to study the infection of the intestinal nematodes in the children, 406 children's faeces were collected and checked in the laboratory. We floated the eggs by saturated NaCl solution and counted the number of the eggs under the microscope. And the pinworm's eggs were collected by transparent adhesive tape. The results showed that the rate of the intestinal nematode infection is 13.79%, all are ascarid. The rate of pinworm infection is 8.87%. The rate of the ascarid's infection in children inside Xiamen Island is 11.96%, for the rate of infection in children of outside the island is 15.32%. The rates of infection varied in different kindergarten. For the rate of ascarid infection, the lowest is 5.00%, the highest is 26.67%; for the rate of pinworm infection, the lowest is 0.00%, the highest is 25.93%. For the rate of ascarid infection, the boys is 12.24%, and the girls is 15.98%; for the rate of pinworm infection, the boys is 6.67%, and the girls is 12.24%, the rate of female is higher than that of male. It concludes that ascarid and pinworm infection are still the important intestinal nematodes for the children in China,but the rate of infection is low in Xiamen.30 dogs faces were collected and checked in the laboratory, the eggs were floated by saturated NaCl solution and incubated on the"T"filter paper. One dog has found infected with Ancylostoma caninum. The results indicate that the infection rate of Ancylostoma caninum is 3.33%, and Ascaris lumbricoides is 10.00% in dogs,respectively. The experimental dogs were infected by infective larvae from skin and mouth. The parasites were collected from small intestine at different time of post-infection and observed via microscope. And the different stages of Ancylostoma canium were observed.SDS-PAGE was used to analyze the protein expression of L3, L4 and adults of Ancylostoma canium. The results show that L3 has 22 bands, L4 has 17 bands, male and female also have 22 bands. The L3 has more protein bands than L4, the reasons may be that L3 is infective larva, and secretes more active proteins and enzymes in order to penetrate the skins of hosts and escape the immune system. There are many different bands between larvae and adults, whereas there are high similar bands between male and female, the reason maybe that the larva must pass through molting and germline development during developing into the adults. Western blot was done by using antiserum of adult worm, the results showed that they both have 40KDa protein band with strong immunogenicity.This study would be applied in the immunology and molecular mechanism of Ancylostoma caninum infection.Two proteinase genes, the aspartic protease (Asp) and cathepsin B protease (AcaB) from Ancylostoma caninum were cloned and expressed in E.coli. In order to provide the foundation of the diagnosis of Ancylostoma as well as for the development of anti-hookworm vaccine, the genes encoding protease Asp and AcaB were amplified from the total RNA by using RT-PCR. The cDNA of asp is about 1200bp and cDNA of acaB is 800bp. The amplified cDNA products were cloned initially into pMD18-T vector and then into expression vector of pET-32a and pGEX-4T-3 respectively, then transformed into E.coli DH5аstrain and cultured on LB plus ampicillin (100μg/ml) plates. Colonies containing the recombinations were selected by PCR and the plasmids DNA were extracted and digested with enzymes. Plasmids containing the right insert were sequenced to confirm their identities, and then the right recombinants were retransformed into E.coli BL21(DE3)strain. Bacterial lysates from cultures induced with IPTG (1mmol/L) were directly loaded onto SDS-PAGE gel, and the proteins on the SDS-PAGE gel were transfered to nitrocellulose membrane, and detected with antiserums against Asp and AcaB respectively. Through these procedures, a specific protein with a molecular mass of 40KDa in Asp western blot could be visualized on membrane, and in AcaB western blot, 30KDa band could be visualized on membrane. The results show that these proteins are expressed in L3, male and female adult worms. These proteins can be used for further studies in the detection of the effectiveness of immunity and the preparation of antigen and antibodies of Asp and AcaB in large scale.