Adh Missing Tags And Diagnosis Of Brucella Live Attenuated Vaccine Strain

Brucellosis, a worldwide zoonotic disease, caused by Brucella, has been seriously restricting the development of animal husbandry and was classified as B infectious diseases in China. Based on host preference, Brucella is divided into six species, namely Malta brucellosis (Br. Melitensis), abortion brucellosis (Br. Abortus), swine brucellosis (Br. Suis), sheep brucellosis (Br. Ovis), sarin mouse brucellosis (Br. Neotomae) and dog brucellosis (Br. Canis). Currently, the most effective way of prevention against brucellosis is vaccination, and the vaccines currently used mainly are live attenuated vaccines, inactivated vaccines and subunit vaccines. However, inactivated vaccines have many disadvantages, like incapability of being replicated in animal body, the need of a large inoculum size, a shorter immune period than live attenuated vaccines, and necessary addition of the appropriate immune adjuvant; subunit vaccine have a higher production cost than live attenuated vaccines. So the vaccine most widely used to prevent brucellosis is live attenuated vaccine. The live attenuated vaccines in market mainly are A19, S2, M5,104M, Rev.1, RB51and so on.Although good results have been achieved in brucellosis prevention and control by using the live attenuated vaccine, there are still some problems. For example, live attenuated vaccine may cause infection after vaccination as its strong toxicity; and if vaccinated animals suffer from brucellosis, it is difficulty to determine whether the infection is caused by vaccine strain or wild. In response to the disadvantages of the live attenuated vaccine, we knock out adh gene at the live attenuated vaccine,so it decreased virulenec and expression of adh into protein,using methods of differential diagnosis of immune serum study infection and nature infection,the following experiments:Experiment1Construction of adh gene deletion mutants of vaccine strains S2, M5and A19First, we selected virulence-associated gene adh, which is a surface antigen gene. by employing DNA microarray and protein microarray. Although its role in the pathogenesis of brucellosis has not been reported, we found that its protein adh is of antigen diagnosis from our results of protein microarray. Then, we constructed the adh gene deletion mutants of S2,M5and A19by kanamycin resistance cassette (kan) replacement:first, amplified the N and C homologous arms of adh gene and kan gene and fused them three into a mutation box with kan gene between the N and C homologous arm; then, the mutation box was ligated to pMD19-T Vector (for simple, as T vector below) as the mutation vector; finally, transformed the mutation vector into compentent cells of vaccine strains S2,M5and A19and selected Brucella transconjugants.Experiment2Analysis for immunoprotection of adh gene deletion mutants of vaccine strains S2, M5and A19First, we analyzed the residual virulence of the adh gene mutants compared to that of the parent strains in Balb/C mice. Balb/C mice were injected by the intraperitoneal route with the same amount of adh gene mutants or their parent strains. Mice were killed at1,2,3,4,5weeks after the challenge. Their spleens were homogenized in PBS, serially diluted, and plated. The results showed that the adh gene mutants were still existed but lower than their parent strains, which suggested a decreased virulence of the adh gene mutants compared to that of the parent strains. Then, we evaluated the protection conferred by the vaccine strains S2, M5and A19with adh gene deletions in Balb/c mice against the B.16M challenge. Balb/C mice were injected with the same amount of adh gene mutants or their parent strains by the intraperitoneal route.5weeks later, the B.16M challenge strain was administered by the intraperitoneal route. Mice from each group were randomly killed by cervical dislocation to isolate the spleens,2,4or6weeks postchallenge. Their spleens were homogenized in PBS, serially diluted, and plated. The results showed that the adh gene mutants induced a higher but not significant protection against the B.16M challenge compared to their parent strains (P>0.05), and coube only be a reference clinically.Experiment3Expression and purification of adh proteinFirst, we used the prokaryotic expression system to express the adh gene into protein. Since the early laboratory work has subcloned adh genes into E. coli2566, only the rest of the expression and purification of adh protein were needed:cultured the E. coli2566containing expression vector in liquid medium LB (Luria bertani medium) to the logarithmic phase followed by adding IPTG to induce protein expression at low temperature and low speed. Then, collected the bacteria after induction and lysed them by sonication to release the protein. Protein was run on SDS-PAGE and Coomassie blue stained to determine whether the target protein expression is successful or not. After this, re-collected cell protein, run them on SDS-PAGE, and according to the molecular weight cut the corresponding bands, then broken them and add protein leaching solution at4℃overnight for precipitation of protein. Finally, measured the concentration of the protein, run them on SDS-PAGE and stained them for verification. The results showed that we successfully expressed and purified the adh protein and provide the antigen for the differential diagnosis in the next experiment.Experiment4Differential diagnosis of adh gene deletion mutants of vaccine strains S2, M5and A19, their parent strains and wild stainsIn this experiment, we used enzyme-linked immunosorbent assay to distinguish among the adh gene deletion mutants, their parent strains and wild stains. The adh gene deletion mutants can not express adh antigen because of the deletion of the adh gene, so mice challenged with adh gene deletion mutants can not produce the antidody against adh antigen. First, Balb/C mice were injected with the same amount of adh gene mutants or their parent strains or wild stains by the intraperitoneal route. Mice were bled at1,2and4weeks after the challenge and get the serum through precipitation. Then, enzyme-linked immunosorbent assays were carried out using the the purified adh protein as antigen and the serum as antibody. The results showed that all the groups were not distinguished well at1week after the challenge (might due to the antibody produced mostly were IgM) and the antibodies produced by the adh gene deletion mutants of vaccine strains S2, M5and A19were far less than those by their parent strains and wild stains, both with a significant difference (P<0.05). Therefore, the results above showed that the adh gene deletion mutants of vaccine strains could distinguish vaccine strains and wild stains.