| Classical swine fever (CSF) is a highly contagious viral disease of pigs caused by Classical swine fever virus (CSFV), and the mortality rate is often very high. Great loss for worldwide development and trade of stock rising has been generated from prevalence of classical swine fever. CSF is included in the list A by the Office International des Epizooties (OIE), and classified as the 1st animal disease in China. Vaccination is the major method to prevent disease. Hog cholera lapinized virus (HCLV) vaccine has been used for a long time and played a key role in the control of epizootic CSF. However, an atypical form of CSF has emerged in China since the 1970s, especially in the recent years, and a major shortcoming of the existing vaccines is lack of marker status, and vaccinated pigs cannot be distinguished from animals infected with field virus. Therefore, developing more efficacious and safe vaccines based on molecular biology and serology is extremely significant in control of this diseases. In the present study, three recombinant adenoviruses pAd-E0, pAd-E2 and pAd-E0-E2 were constructed which could express E0 and/or E2 antigen of Classical swine fever virus Shimen strain in vitro, and the protection effect of these recombinant adenoviruses were investigated, and offered some technology exploration for live vector vaccines of CSFV.1. Construction and identification of pAd-E0, pAd-E2 and pAd-E0-E2 which expressing CSFV Shimen strain E0/E2 proteins. The E0 gene and E2 gene fragments of CSFV Shimen strain were amplified from pMD-18T-E0 and pMD-18T-E2 vectors by PCR with specific primers, and cloned into adenovirus shuttle vector pAdTrack-CMV respectively to produce recombinant adenovirus shuttle vectors: pAdTrack-E0 and pAdTrack-E2. The E0 gene and E2 gene fragments were seriated in pET-32a vector. E0-E2 fragment was obtained by use Kpn I and Not I to digest the vector, and E0-E2 gene fragment was inserted into adenovirus shuttle vector pAdTrack-CMV also, and the vector was named pAdTrack-E0-E2. pAdTrack-E0, pAdTrack-E2 and pAdTrack-E0-E2 vectors were linearized by Pme I restriction enzyme. After these three kinds of linearized adenovirus shuttle vectors were recycled, they were transferred into E.coli BJ5183 cells which contained adenoviral backbone plasmid pAdEasier-1. The recombinant adenoviral backbone plasmids which were named pAdEasy-E0, pAdEasy-E2 and pAdEasy-E0-E2 were generated in BJ5183 by homologous recombinant machinery presented in bacteria. After digested by Pac I, pAdEasy-E0, pAdEasy-E2 and pAdEasy-E0-E2 were transfected into HEK293 cells respectively. Three recombinant adenoviruses: pAd-E0, pAd-E2 and pAd-E0-E2 were propagated in 293 cells. The control recombinant adenovirus pAd-CMV was constructed with the same method. The classical cytopathic effects (CPE) could be observed of these three recombinant adenoviruses. pAd-E0, pAd-E2 and pAd-E0-E2 could carry and express E0, E2 and E0-E2 proteins respectively by identification of PCR and Western blot. The three recombinant adenoviruses had good genetics stability by 10 times subculturing test.2. The immune efficacy of the three recombinant adenoviruses was evaluated in mice. 12 BALB/c mice were divided into three groups (4 mice in each group), and inoculated intraperitoneally two times at a two-week interval with 1×108 pfu of amplified and purified pAd-E0, pAd-E2 and pAd-E0-E2 respectively. Blood was collected from the beginning of inoculation. The detection of antibodies against CSFV were performed using ELISA antibody detection kit. After the second immunization, all groups developed CSFV specific antibody. The highest titers of serum anti-CSFV were 1:10 240, 1:20 480 and 1:10 240 respectively. It provided a foundation for further study on the protection of theses three recombinant adenoviruses on pigs.3. Evaluation on the protection of pAd-E0, pAd-E2 and pAd-E0-E2. The commercially available pig's susceptibility to CSFV was determined 21 days after twice immunizations through subcutaneous and intramuscular routes by three recombinant adenoviruses which contained the Classical Swine Fever Virus (CSFV) E0 and/or E2 gene, and set pigs inoculated non-recombinant adenovirus and DMEM as negative control. The protections of pigs vaccinated with pAd-E0, pAd-E2 and pAd-E0-E2 were 50 percent (2/4), 75 percent (3/4) and 75 percent (6/8). 60 percent of animals (3/5) vaccinated with commercial vaccine were protected, and all animals vaccinated with the non-recombinant human adenovirus (4/4) and blanks (2/2) dead. Before challenge, low antibodies to CSFV were detected in all pigs vaccinated with recombinant adenoviruses. High antibodies were detected in all animals vaccinated with commercial vaccine, but none in blanks. The number of pigs had a temperature above 40℃(indicative of fever) were tested every day after challenge. 75 percent of animals (3/4) vaccinated with pAd-E0, 50 percent of animals (2/4) vaccinated with pAd-E2, and 50 percent of animals (4/8) vaccinated with pAd-E0-E2 developed temperatures were above 40℃, 80 percent of pigs (4/5) vaccinated with commercial vaccine developed temperatures were above 40℃, and did all blanks developed temperatures were above 40℃, The analysis of clinical symptom showed that the recombinant adenoviruses can offered protection to CSFV. The results showed that the recombinant adenovirus contained CSFV E0 gene could offer less protection for pigs than commercial vaccine, but recombinant adenoviruses contained CSFV E2 could offer more protection than commercial vaccine.
|
PDF Full Text Request