Studues On The Coexpression In E.Coliand The Immune Regulation Of Avain Influenza H9N2Inactived Virus Of Thymosin Alpha1-Bursin Like Peptide

The tripeptide bursin (BS) Lys-His-Gly-NH2was first isolated from bura of Fabricius. And it is a biological response modifier with multiple biological activities. It has the ability to induce B cell differentiation, stimulate stem cell differentiation and promote the switching from IgM to IgG.It therefore has high potential as an adjuvant. The previous study reported that "T-X-N-L" like peptide perhaps was the conservative sequence binding to anti-bursin monoclonal antibody (2F9-4/HU2). The TXNL-like peptide also showed a similar effect on stimulating the antibody in comparison to bursin in bioactive tests. The previous study shown that tandem TXNL-like peptide and bursin fusion peptide (TPNL-KHG, designated as bursin like peptide, BLP), is a potent enhancer of immune response in mice immunized with the JEV subunit vaccine, and represents a promising adjuvant for vaccination.Thymosin alpha1(Tal)(Zadaxin, SciClone Pharmaceuticals, San Mateo, Calif), a highly conserved28amino acid peptide was initially isolated from the Thymus gland. It has abilities to stimulate the T-cells maturation, differentiation, and affect the function of T-cells, increase the efficiency of antigen presentation by macrophages, promote DC differentiation, which are the most effective antigen presenting cells and play an important role in immune response. Tal was approved in South Korea in April2000, as an influenza vaccine adjuvant.Since Tα1and BLP have the potent adjuvant effects, we designed and synthesized thymosin α1-bursin like fusion peptide, and used it as adjuvants to test if it can induce immune responses in mice upon H9N2avian influenza virus (WIV) vaccination. All studies were listed as below:1.Design and expression of the thymosin a1-bursin like peptide gene in E.coli. Based on the amino acid sequence of the Thymosin al (DAAVDTSSEITTKDLKEKKEVVEEAEN) and the bursin like peptide (TPNLKHG), we designed the tandem peptide Thymosin al-bursin like peptide (Tal-BLP). The amino acid sequence is GGGGS-SDAAVDTSSEITTKDLKEKKEVVEEAEN-GGGGS-TPNLKHG, with GGGGS as linker. Based on codon usage of E. coli, peptide coding sequences were designed and synthesized using gene splicing by overlap extension PCR (SOE-PCR) method.The156bp synthetic thymosin al-bursin-like peptide(Tal-BLP), with an upstream EcoRI site and a downstream Xho site, was cloned into pET-32a(+) expression vector to produce the thioredoxin-Thymosinal-bursin like peptide(TRX-Tal-BLP). All the plasmids used in this paper were propagated in the E. coli. strain DH5a. Recombinant proteins of TRX-Tal-BLP, TRX-BLP and TRX pET32a(+) vector alone were expressed in E. coli BL21. All three recombinant proteins were expressed as a soluble protein. The supernatant was purified on a Ni-affinity chromatography column. This study is the basic for further study.2.Influenza inactive virus with thymosin al-bursin like peptide enhances the immunity of mice.Adjuvant play an important role in the formation of effective and appropriate vaccines. As both bursin-like peptide (BLP) and Thymosin al (Tal) are potent adjuvants, the tandem peptide of Thymosin al-bursin like peptide (Tαl-BLP) was synthesized. To test whether the Tal-BLP was potent adjuvant that could enhance specific immune responses, the whole-inactivated avian influenza virus (WIV) was used as immunogen combined with different concentrations of Tal-BLP or BLP. The effect was determined in the form of protective HI, anti-WIV H9N2AIV titers, antibody isotypes (IgGl and IgG2a), spleen cell lymphocyte proliferation by using MTT assay as well as Thl (IFN-y) and Th2(IL-4) cytokines production. To assess the effects of adjuvants on viral clearance from the lungs, mice were challenged intranasally under anaesthesia with H9N2AIV2weeks later, and the virus titers in lungs were detected by Real time RT-PCR. Our data represented in this paper shown that the use of tandem peptide Tal-BLP as the adjuvant strongly enhanced the antibody titers and the HI titers. And the titers of the groups that co-immunization WIV with Tal-BLP were higher than the other groups after the prime and first immunization. The results demonstrated that adding the Tal-BLP can switch the immune response to WIV from a predominantly Thl type to a mixed Thl/Th2one. And using medium dose of Tal-BLP as adjuvant is beneficial for the clearance of H9N2AIV in the lungs. Thus Tα1-BLP represents a promising adjuvant for H9N2avian influenza vaccination. And it may be a promising adjuvant for human vaccine applications.