| The native JL94 isolate of porcine rotavirus (RV) was propagated in MAl04 cel1 culture,and harvested after 48 hours. Double-stranded RNA extracted from the virus was used as thetemplate for reverse transcription. Using two pairs of primers (Li-9 and Li-11, Li-12 andLi-13), two gene segments were amplified by reverse transcription-polymerase chain reaction(RT-PCR), respectively. The two gene segments were inserted into pMD18-T vector andidentified by sequencing, single and double digestion with restriction endonucleases,respectively. One of the two gene segments was gene encoding oater capsid protein VP7,another was gene encoding inter shell protein VP6. The two recombinant plasmids namedpMD18-T-JL94/VP7 and pMD18-T-JL94/VP6 were constructed. The result of their homologycomparison of nucleotide sequence with OSU/VP7, Gottfried /VP7 is 99.9%, 74.1%,respectively; with OSU/VP6, Gottfried/VP6 is 86.8%, 82.4%. The result of their homologycomparison of amino acid sequence with OSU/VP7, Gottfried /VP7 is 99.9%, 75.4%, withOSU/VP6, Gottfried/VP6 is 97.5%, 93.2%.The gene of VP7 containing Kozak's sequence was amplified from pMD18-T-JL94/VP7by PCR using a pair of prirner (Li-l4 and Li-11) designed according to the sequence ofJL94/VP7 gene cDNA, then it was inserted into pMD18-T vector and was identified bydigestion. The recombinant plasmid was pMD18-T-VP7. The VP7 gene from pMD18-T-VP7digested by restriction endonucleases was recombinated into eukaryotic expression plasmidpcDNA3.1(+). The recombinant plasmid, pcDNA-VP7, was constructed successfully afterbeing identified by sequencing and digestion.The transmissible gastroenteritis virus (TGEV)TH98/N gene was amplified with a pair ofprimer (Li-l5 and Li-2)from the recombinant plasmid, PHD. After being digested, it wasrecombinated inio euknyotic expression plasmid pcDNA3. 1 (+). and named pcDNA-N. The Kunming mice used for nucleic acid immtalzation were 6 to 8 weeks of age anddivided into four groups (35 mice each). Group A was injected with 100 μg of pcDNA-VP7,group B with 100μg of pcDNA-N, group C with 100μg of pcDNA-VP7 and 100μg ofpcDNA-N, group D with 100μg of pcDNA3.1(+) in 100μg l volume via femoral quadricepsintramuscular route, respectively, and the mice in ereh group were injected be times totallyAfter primary injection, all mice received booster injechon two thoes with the sazne dosageand via the sarne route of primny injection at interval of two weeks. Blood saznples collected3from three mice at 0d before primary injection and those colIected from three mice of eachgroup at l4d, 2ld, 28d, 35d. 39d, 47d, 54d, were used for detecting antibody levels and thechanges of CD4+, CD8+ T cells in peripheral blood lyInphocytes.The resultS showed that the anibodies against rotavirus JL94 couId be deteCted in theserum of mice from grouP A at l4, 28, 35, 39 and 54 days of POstimmMon (PD, and thesame anibodies also could be detected in the serum of mice from group C at l4, 28, 39 and 54days of PI. The anibodies to N protein of TGEV could be detected in the serum of mice frOmgroup B at 39 days of PI.There were significant differences rert0.05) in number of CD4+ T cells in Peripheralblood lymphocytes between group A and group D at l4, 47 and 54 days of PI, group A washigher than group D. There were sighficant differences (Prt0.05) in number of CD4+ T cel1sin peripheral blood lymphocytes betWeen group B and group D at 28 and 47 days of PI, grOuPB was higher than group D. There were significant differences (Pwt0.05) in number of CD4+ TceIls in peripheraI blood lymphocytes betWeen group C and group D at 28 days of PI, group Cwas higher than grOup D.There were significant differences (Prt0.05) in number of CD8+ T cells in PeripheralbIood Iymphocytes betWeen group A and group D at l4 days of PI, group A was nigher thangroup D. There were significant differences (P<0.05) in number of CD8+ T cells in Peripheralb1ood lymphocytes b... |
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