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Fusion albumen containing HSP70 ATPase structure field and antigen peptide and application thereof

A fusion protein and domain technology, applied in the field of fusion proteins, can solve the problems of low expression of fusion proteins, difficult industrial production scale, and amplification, and achieve the effects of less adverse reactions and good immune effects.

Inactive Publication Date: 2007-02-28
BEIJING SUNBIO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the expression level of fusion proteins formed by full-length Hsp70 and tumor antigen peptides or pathogen antigens is very low, and it is difficult to scale up industrial production

Method used

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  • Fusion albumen containing HSP70 ATPase structure field and antigen peptide and application thereof
  • Fusion albumen containing HSP70 ATPase structure field and antigen peptide and application thereof
  • Fusion albumen containing HSP70 ATPase structure field and antigen peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, recombinant human HSP70 ATPase domain and HPV E7 fusion protein (AE7) coding

[0036] Gene construction and expression

[0037] The E7 and E6 proteins of human papillomavirus (Human papillomavirus, HPV) play an important role in the tumorigenesis of HPV-infected patients (Fehrmann and Laimins, 2003). Therefore, the E7 and E6 proteins on the cell surface of HPV-infected patients can serve as tumor antigens. recognized by T cells. The fusion protein composed of the ATPase domain of Hsp70 and E7 / E6 can enhance the immune response of the human body to HPV virus and the cervical cancer caused by it. We named the fusion protein of ATPase domain of Hsp70 and E7 as AE7, and the fusion protein of ATPase domain of Hsp70 and E6 as AE6.

[0038] The human Hsp70 ATPase domain gene (amino acid residues 1-386) was obtained from the full-length human Hsp70 gene by PCR. HPV virus type 16 E7 gene was obtained from the virus genome PCR. The Hsp70ATPase do...

Embodiment 2

[0044] Embodiment 2, recombinant human HSP70 ATPase domain and HPV E6 fusion protein (AE6) coding gene

[0045] construction and expression

[0046] The HPV virus type 16 E6 gene was obtained from the virus genome PCR, and the PCR product was digested with restriction endonucleases BamH I and Xho I and connected to S28bA, named S28bAE6, and the nucleic acid sequence of AE6 was confirmed by sequencing.

[0047] S28bAE6 plasmid was transformed into E coli BL21(DE3) strain for protein expression. The expression method of AE6 protein is the same as that of AE7. The refolding and purification of AE6 protein is similar to that of AE7. The ATPase activity of AE6 was detected by malachite green method. The results showed that the recombinant AE6 had all the functions of hydrolyzing ATP.

Embodiment 3

[0048] Example 3, recombinant human HSP70 ATPase domain and human p53 fusion protein (hAP53) encoding gene

[0049] construction and expression

[0050] The human p53 gene was obtained by PCR using the human liver cDNA library as a template. The PCR product of human p53 gene was digested with restriction endonuclease and connected to S28bA, named as S28bhAP53, and the nucleic acid sequence of the target gene was confirmed by DNA sequencing.

[0051] The protein expression, refolding and purification methods of hAP53 were similar to those of AE7, and the ATPase activity was detected by the malachite green method. The results showed that the recombinant hAP53 had all the functions of hydrolyzing ATP.

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Abstract

The invention discloses a framework area including human HSP70 ATPase and human papillomavirus E7, E6 protein or merge protein of mammalian p53 protein. The invention also discloses a pharmaceutical compound of merge protein, gene of encoding merge protein, the expressive carrier of merge protein and the usage of preparing medicine to treat or avoid tumor as human papillomavirus or tumor related to p53 protein overdose express. The invention also discloses a pharmaceutical compound of merge protein, gene of encoding merge protein, the expressive carrier of merge protein and the usage of preparing medicine to treat or avoid tumor as human papillomavirus or tumor related to p53 protein overdose express.

Description

technical field [0001] The invention relates to a fusion protein comprising an adenosine triphosphate (ATPase) domain of a heat shock protein (hereinafter referred to as ATPase) domain and an antigenic peptide, more specifically, the invention relates to a fusion protein comprising a human HSP70 ATPase domain and human papillomavirus E7, E6 protein or Fusion protein of mammalian p53 protein. The present invention also relates to a pharmaceutical composition comprising the above-mentioned fusion protein, a gene encoding the above-mentioned fusion protein, an expression vector comprising the gene and its preparation for treating or preventing human papillomavirus-related tumors or p53 protein overexpression. Use in medicine for tumors. Background technique [0002] It was discovered long ago that heat shock protein (heat shock protein, Hsp) can promote the correct folding of protein molecules. Recently, it was found that Hsp70, Hsp90 and Grp94 (also known as gp96) can submit...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/16A61K38/43C12N15/62C12N15/63A61P35/00
Inventor 朱冰
Owner BEIJING SUNBIO BIOTECH
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