Recombinant protein replacing GLS epitope and encoding nucleotide thereof

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The invention belongs to the field of bio-pharmacy, and particularly relates to GPC3-HBsAg recombinant protein and application thereof in a tumor vaccine. The recombinant protein is recombined hepatitis B surface antigen (HBsAg) and specifically is recombinant protein of which the HBsAg endogenous CTL (Ctotoxicity T Cell) epitope is replaced by another exogenous CTL epitope; the exogenous CTL epitope is CTL epitope of GPC3.

The present invention belongs to the field of bio-medicine, in particular to GPC3-HBsAg recombinant protein and its encoding nucleotide and applications.

Background technique:phosphatidylinositol proteoglycan (glypican) family are present in the new family of heparan sulfate proteoglycans on the cell surface. Been reported so far five phosphatidyl glypican (glypican 1 phosphatidylinositol, phosphatidyl glypican 2, glypican 3 phosphatidylinositol, phosphatidylinositol 4 proteoglycan, and phosphatidyl glypican 5) is a member of the phosphatidylinositol proteoglycan family. Members of the family have a uniform size (about 60kDa) core protein, there are specific, highly conserved cysteine ​​sequences, binding and anchored by glycosyl phosphatidylinositol (GPI) to the cell membrane.

Motomura et al found that GPC3 can be induced in mice GPC3-specific anti-tumor immunity, but not in mice GPC3 cause autoimmune diseases. At the same time it found that a GPC3-specific killer T cell (CTL) can contribute to the occurrence of the expression of tumor tissue GPC3 orientation Gan shift without causing autoimmune destruction. Therefore, GPC3-specific CTL induced by the directional movement of anti-tumor immunity can be used as a possible means of HCC and melanoma treatment. And CTL epitopes can be identified in mice CTL and induce specific cellular immunity, to the discovery of GPC3 as a target for cancer immunotherapy has important significance confirmed by experiment.

HBsAg vaccine and compared to conventional vaccine has the following advantages: 1 without immunological adjuvant that is able to induce efficient and specific cellular and humoral immunity; 2.HBsAg itself can not be copied, and no infection; 3. HBsAg can be a large number of in vitro amplification, and tend to concentrate and purify. These characteristics show, HBsAg having a live attenuated virus vaccine immunogenicity but not its shortcomings. Other than in a specific exogenous antigen (gene) is inserted HBsAg or HBsAg alternative source CTL epitopes on endogenous specific CTL epitopes in vivo can induce efficient and specific cell-mediated immunity, vaccine research has increasingly become a hot spot . It has been shown effective in retaining its packaging epitopes on the basis of the HBsAg gene open reading frame (open reading frame, 0RF) mutagenesis out endogenous CTL epitopes and establish endonuclease cleavage site Construction good recombinant plasmid allows the insertion of one or more foreign epitopes, HBsAg retain some biological and immunological properties of the basis, but also to ensure that exogenous immunoreactive epitopes.

Non-Patent Document Wang Wenjing, etc., GPC-3CTL epitope expression vector, Southern Medical University, 2009, 29 (8), 1548-1550; discloses a construct encoding GPC3-HBsAg recombinant protein expression vector using GPC3 CTL epitopes are replaced with HBsAg in FLG, LLD and SIL epitopes to obtain an expression vector, and a certain immunogenicity. However, the presence of HBsAg in the art a plurality of CTL epitopes, epitope different replacement immunogenicity quite different, so choosing the right HBsAg CTL epitopes for replacement, the obtained recombinant immunogenic protein decisive Impact.

In a preferred embodiment of the present invention, the recombinant hepatitis B surface antigen (HBsAg), one, two, three or more endogenous HBsAg CTL epitopes are one, two, three or more CTL epitopes of GPC3 replaced.

In another embodiment of the present invention, the amino acid sequence of the recombinant hepatitis B surface antigen (HBsAg), such as SEQ ID N0: 1 as shown; in the SEQ ID N0: 1 in the sequence, of HBsAg endogenous CTL epitopes correspondingly GPC3 CTL epitopes replaced.