| A strategy to enhance immune responses to conventional vaccines and DNA vaccines is very important in further development of the vaccine to control animal infectious diseases. The immunogenicity of vaccines can be significantly elevated with the use of powerful adjuvant. With the development of molecule biology and immunology, cytokines have been used as immunoadjuvants. Although cytokine can strongly trigger humoral and cellular immune responses to certain antigens or pathogens, main disadvantages of cytokines as adjuvant include high cost, difficult to produce and purify, short-lived bioactivity, and the need for frequent boosts. Recent studies have shown that cytokine gene adjuvant, CpG motifs could stimulate immunogenicity of vaccine and increase levels of immune responses.Interleukin-4 (IL-4) is a versatile cytokine that plays an important role in the regulation of immune responses. It is engaged in regulating the biological activities, such as interleukin gene expression, cell proliferation, B cell differentiation, T cell activation, and neutrophil and macrophage differentiation.Now few are known about the effect of porcine interleukin 4 on the immunity and the immune responses to vaccination. Furthermore, there is still lack of systemic explorations on the effect of transgenetic expression of swine IL-4 gene in vitro and vivo, on the immune responses of animals induced by routine protein vaccination. Our experiment is unique to clone porcine IL-4 gene from lymphocytes of the local species of Chenghua porcine and systematically study its expressing effect in vivo on the immune system and immune responses of mouse and pig to trivalent vaccines against Swine fever, the Pasteurellosis and Erysipelas suis and pig paratyphoid vaccine. It is expected to develop a novel immune potentiator to increase the immunity and enhance the immune responsesof porcine to various vaccines and elevate the immunoprotective efficacy against different infectious pathogens.Porcine interleukin-4 cDNA from lymphocyte was synthesized by RT-PCR from mRNA extracted from blood lymphocytes of Chenghua pig, which were stimulated by lOug/ml Con A in vitro. The porcine interleukin-4 cDNA from lymphocyte was then subcloned into pMD18-T vector and was named as pUPIL-4. The sequence of cloned porcine interleukin-4 cDNA was determined. The length of the IL-4 gene was 413 bp, encoding a peptide of 134 amino acids whose molecular weight is 15.0 KDa and pi is 8.24 . By blasting the homologous sequences in GenBank databases, the homology of interleukin-4 amino acids sequence from lymphocyte between Chenghua porcine and Large White porcine is 99%; the homology between Chenghua porcine and hybrid breed pig is 96%. The sequence of pig IL-4 gene from lymphocyte was submitted to GenBank and registered as AF493991.The eukaryotic expression plasmid was constructed and named VPIL-4. The ability of VPIL-4 to express full-length porcine interleukin-4 in vitro was assured by RT-PCR in Cos? cells that were transiently transfected with VPIL-4 gene entrapped with Lipofectamine and by the biological activities of the expression products to increase remarkably the proliferation of lymphoblast from the blood of porcine and from spleen of mice stimulated by Con A .270 BALB/c female mice at the age of 6 weeks were divided into 18 groups and were intramuscularly vaccinated respectively with 30 pmol of naked VPIL-4 and pUC18-CpG, 6 pmol of VPIL-4 and pUC18-CpG entrapped with cationic liposome or PLG nanometer particles prepared in our lab, and some of the mice were immunized with the trivalent vaccines and pig paratyphoid vaccine. Mice were bled on 0, 7, 14 and 21 days after immunization to determine the dynamic changes of white blood cells, the specific antibody titres and the content of IgG; the spleen were collected from the mice to detect the proliferation and the bioactivities of the induced interleukin-2 of lymphocytes to stimulation with Con A by MTT assay. The experimental results indicated that the number of white blood cells significantly increa...
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