| Classical swine fever(CSF), caused by Classical swine fever virus(CSFV), is a highly contagious desease with the characters of fever, generalized haemorrhages and ichoremia, and leads to devastating economic losses during outbreaks, which can infect all kinds of swine. In recent years, there were many reports about the immunological failure of CSF. The popularity of CSFV presents some new characters, which is related to the CSFV variation by high immunological pressure. So, in order to control CSFV effectively and reduce the economic losses, it is essential to carry out an epidemiological survey of CSF for better understanding of the epidemiological status of CSF and the genetic mutation of CSFV, and research on the subunit vaccine for eradicaiton of CSF.The IDEXX CSFV antibody and antigen test kit were used to detect the level of antibody and antigen of1411sera samples from10pig farms, in which1028sera samples from these10pig farms were used to detect the antibody level, others from5pig farms with lower immunity level of antibody among these10nurseries were used to detect the antigen infection. The results showed that the antibody positive rates against CSFV are45.34%-84.00%, and the antigen positive rates of CSFV antigen reach0%-2.32%. It means that the immunological status of CSF in the survillianced area of Shandong province is sophisticated, Some of the pig farms show a low immunity level, and carries CSFV.In this study, a total of13samples from10pig farms referred above in2011were collected and detected by reverse transcriptase polymerase chain reaction(RT-PCR). Six samples were found to be positive, which were inocalated in PK-15cells. As a result, six CSFV strains were isolated and were named as HZ,LW,YT,JN,RZ,TA respectively. The real time RT-PCR was used to certify that what we got all belonged to the epidemic field strains. The results futher conformed that the immunized pig farms really carried CSFV. So, In order to prevent from infection of CSF, the antibody against CSFV should be surveillanced regularly and appropriate measures be carried out.According to the results of the comparison and analysis of E2gene among the isolated strains and reported nucleotide sequences on GenBank by DNAStar software, we found that it was highly homogeneous among the six separated strains. When compared the6strains with other classical strains such as Shimen stain, HCLV strain, GuangXi strain and so on, the results showed that the isolated strains and GuangXi strain shared so high homology in nucleotide and amino acid sequence. However, the homology between the isolated strains and Shimen strain was81.3%-84.3%for nucleotide sequence and84.4%-89.2%for deduced amino acid sequence, and compared the isolated strains with HCLV strain they shared79.4%-82.3%in nucleotide sequence and83.2%-88.0%identities in amino acid sequence respectively. The results indicated that the isolated strains perhaps have a close relationship to the CSFV strains in Guangxi province. The prevalent CSFV strains in Shandong have been evolving slowly to the direction away from the HCLV vaccine strain. and The phylogenetic tree shows that the six separated strains and GuangXi strain belong to genetic group â…¡ and also Subgroup2.2, otherwise, the Shimen stain and HCLV strain belong to genetic group I.Finally, one pair of primers was designed to amplify the E2gene without TMR which encode the envelope glycoprotein, and the PCR products were inserted into secretory Pichia. pastoris expression vector pPICZaA. Then the vector was linearised and transformed into Pichia.pastoris X-33by electrotransformation. Transformants with high copies were acquired when selected under Zeocin and were induced with methonl. SDS-PAGE and Western blot analysis showed that the recombinant E2protein was successfully expressed in Pichia.pastoris, which laid the foundation for developing the subunit vaccine. |
