| Swine influenza (SI), caused by swine influenza A virus(SIV), is an acute respiratory disease of pigs industry. The disease can occur all the time during a year, especially in spring and fall. Clinically there were no medicines to cure the sick animals except for preventation by vaccination. Considering the disadvantages of SI inactivated vaccines which contain H1N1 and H3N2 whole virion, researches are trying to develop new types of SI vaccines in order to get the good result of vaccination.According to the sequence of SIV hemagglutination (HA) and Neuramidase(NA) genes, several T and B cell epitopes were deteremined. Here we seleted 3-4 epitopes and ligased by the interval sequence. This chemric gene has the similar antigenecity with the SIV viron. The gene was amplified by PCR and was cloned into pET-30a vector, named pET-30a-EPl. The recombinant plasmids were identified by sequencing and then was transformed into the BL21 (DE3). After induced by 1mM IPTG at 37℃, the recombiant protein was successfully expressed. Themoleuclar weight of recombiant protein EP1 was 27KD in SDS-PAGE. The proteins were purified by affinity chromatography on a nikel-agarose resin. The results of Western blotting revealed the recombinant products could react with the antibody to His-tag, indicating that the recombinant protein was obtained as expected. The concentrations of recombinant proteins were determined to be 1306.88μg/mL.KM mices of 8 weeks ages were immuninzed by EP1 or EP2 protein through subcutaneous injection (s.i), and intranasally (i.n.), respectively. After third immunization, serum and nasal elution were detected. Serum and mucosal antibody levels were detemined by indirect ELISA, HI antibody level of SIV was detected using hemagglutination inhibition (HI), and cell immunity level was measured by MTT. After the mice were immunized for three times, the serum IgG levels maintained at the high level in the EP1 s.i. group and EP2 s.i. group. There were significant differences in serum IgG levels among the EP1 i.m. group and PBS group. But there was no significant difference in serum IgG level between EP2 i.n. group and PBS group (P>0.05). The mucosal IgA antibody was detected in EP1 i.n. group and EP2 i.n. group during the entire immunity process. The results of T lymphocyte transformation assay using MTT method indicated, at 20 days after third immunization, there were significant differences between all the immunized group and control group (P<0.01). The HI experiment indicated that, HI antibody titers to SIV H1 arrived above 1:128 in the s.i. groups of EP1 and EP2 protein and 1:64 in the i.n. groups of EP1, but HI titer was only 1:4 in EP1 i.n. group, which was consistent with ELISA result. It suggested the EP1 and EP2 protein had good immunogenicity. Regardless of the EP1 protein by what way immunity mouse, had the good humoral immunity and cellular immunity response, in which i.n. group had also obtained the ideal partial immunity secretion IgA immune body. HI assay examined the anti-SIV HI antibody, which may suppress red blood cells agglutination activeness of SIV, warrantting the organism eliminate the infection virus. The research obtained recombinant SIV antigen which had been possible to stimulate the cellular immunity, the humoral immunity, the partial mucous membrane immune response. This study established a method for developing the epitope vaccine against SIV subgroups coinfection. |
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