| Swine influenza (SI), caused by influenza A virus, is an acute respiratory disease of pigs. This viral disease is epidemic all the year, but most in spring and fall. Clinically there were no medicines to cure SIV except for vaccination now. Considering the disadvantages of SI inactivated vaccines, which are univalent or double valent with H1N1 and H3N2 whole virus, people are try to develop new types of SI vaccines. SIV entered organisms through the upper respiratory tract mucous membrane to cause infection, Therefore the mucous membrane immunity vaccination was the best immunity way to prevent SIV infections. Heat-labile enterotoxin (LT) of Escherichia coli has some functions on mucosal immunogenicity and adjuvant.Nucleotide sequences and three diamensions structures of SIV hemagglutination (HA) genes were analysed by means of soft, T and B cell epitopes were screened and connected by adaptor, and conjuncted with B subunit of LT (LTB). Digested with NcoI and HindIII, PCR product of the gene with T and B cell epitopes was cloned into the expression vector pET-30a(+), named pET-30a-ep. PCR product of LTB gene was digested with Hind III and XhoI, and further cloned into the expression vector pET-30a-ep, designated the expression vectors pET-30a-EL1 or pET-30a-EL2 respectively according to having stop codon TAA. The recombinant plasmids were identified by sequencing, and transformed into the BL21 (DE3) and induced to express by 1mM IPTG at 37℃. TheExpression products were identified by SDS-PAGE, and found to be 33KD (EL1) and 38KD (EL2) as expected respectively. 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 two recombinant proteins were obtained as expected. The concentrations of recombinant proteins were determined to be 1158.88μg/mL and 1245.90μg/mL by BCA kit, respectively. KM mices of 8 weeks ages were immuninzed by EL1 or EL2 protein through intramuscular inoculation (i.m), 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. On the basis of ELISA square matrix titrate experiment, in order to detect EL1 serum, the optimization condition was EL1 coating antigen in 1: 16 (the 62.5ng/hole), the serum in 1:80, EL1 nosal elution coating antigen in 1: 64 (the 15.6ng/hole), nosal elution 1:3. But for EL2 serum samples, coated antigen with the concentration of 1000ng/hole and 1:160 of serum were best. Similarly, for EL2 nosal elution, coating antigen with 1000ng/hole and 1:3 of nosal elution were best. After the mice were immunized for three times, the serum IgG levels maintained at the high level in the EL1 i.m. group and EL1 s.i. group, and the ELISA antibody titer achieved above 1:10000. There were significant differences in serum IgG levels among the EL1 i.m. group, EL1 s.i. group, EL1 i.n. group and PBS group. But there was no significant difference in serum IgG level between EL1 i.n. group and PBS group (P>0.05). In all immunity groups of EL2 protein serum IgG antibody levels were higher than that of PBS group (P<0.01). Moreover, there was significant difference among the different groups of EL2 protein (P<0.01). The mucosal IgA antibody was not detected in EL1 i.n. group during the entire immunity process. But after third immunization, there was significant difference in mucosal IgA level between EL2 i.n. group and the PBS group (0.01 |
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