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No-adjuvant therapeutic protein vaccine containing heat shock protein and HPV 16Z protein antigen

A technology of heat shock protein and protein antigen, applied in the field of unadjuvanted therapeutic protein vaccine containing heat shock protein and HPV16Z protein antigen, which can solve problems such as lack of adjuvant

Inactive Publication Date: 2007-01-24
张伟
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immunogenicity of pure E6 / E7 fusion protein is not enough to stimulate cellular immune response, and requires the cooperation of adjuvants, but there is still a lack of effective adjuvants approved for use

Method used

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  • No-adjuvant therapeutic protein vaccine containing heat shock protein and HPV 16Z protein antigen
  • No-adjuvant therapeutic protein vaccine containing heat shock protein and HPV 16Z protein antigen
  • No-adjuvant therapeutic protein vaccine containing heat shock protein and HPV 16Z protein antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The construction of embodiment one Hsp65-E6 / E7 fusion protein expression plasmid and bacterial strain

[0017] 1.1 Synthesis of E6 / E7 fusion gene

[0018] Using the whole genome sequence of the HPV16Z virus strain cloned from Shanxi by Professor Zhang Wei as a template, the E6 and E7 fragments were obtained by PCR, and on this basis, 3 pairs of primers were designed to spot the 24, 26, 58, and 91 codons of E7 Mutate, and at the same time PCR amplify the codon of 88 amino acids at the C-terminus of E6, and fuse the obtained part of the E6 fragment with the mutated E7 fragment.

[0019] 1.2 Acquisition of PET28a-Hsp65

[0020] Purchase 2 tubes of BCG from the China Institute of Biological Products, use STE to suspend the bacteria, centrifuge and resuspend in 400μl 0.05MTris-HCl (PH 8.5), 0.05M EDTA, 15% sucrose, add 0.4mg lysozyme, 37℃ Act for 30 minutes, then add 2% proteinase K to a final concentration of 0.1mg / ml, act for 10 minutes at 25°C, add 4mg SD...

Embodiment 2

[0023] Example 2 Identification of fusion protein expression products and optimization of expression conditions

[0024] When the Escherichia coli BL21 (DE3) transformed with the expression plasmid was grown at 37°C in a medium containing kanamycin until the OD600 was between 0.6-1.0, part of the bacterial liquid was taken out, and the rest was added with IPTG to a medium concentration of 1 mmol / L, continue to cultivate at 37°C for 3 hours, take 1ml of the induced and uninduced bacterial solutions, collect the bacterial cells by centrifugation, add the loading buffer in a water bath at 100°C for 5 minutes, centrifuge to get the supernatant and directly perform SDS-PAGE electrophoresis, and also Carry out electrophoresis of protein standard BSA. After electrophoresis, Coomassie brilliant blue staining for more than 2 hours, and then decolorization treatment with decolorization solution, the decolorization process can be accelerated by heating. Compared with BSA, the...

Embodiment 3

[0026] Example 3 Fermentative expression and purification of fusion protein

[0027] After recovering the preserved expression strains, culture them overnight in 10ml LB medium at 37°C with shaking at 150 rpm, inoculate 5ml into 500ml LB medium, and incubate for 24 hours. Carry out fermentation culture in 9.5L TB culture medium, adjust pH value to 6.8, 300 rev / min, culture 4 hours, guarantee that dissolved oxygen is above 50% during this period. Add IPTG with a final concentration of 1 mmol / L, induce for 3 hours, harvest the bacterial liquid, collect the bacterial cells by centrifugation, and suspend them in the lysis buffer A (10 mM Tris-Hcl, 0.5 mM β- mercaptoethanol, pH8.0, 200ug / ml lysozyme), stored at -70°C for later use. When in use, thaw the frozen bacterial suspension at room temperature, add 2ug / ml protease inhibitor PMSF, sonicate the bacteria, centrifuge at 15000rpm for 10 minutes, collect the precipitate, and wash the precipitate with inclusion...

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Abstract

The present invention relates to the fusion protein of heat shock protein and HPV16Z E6 / E7, the obtaining process and expression vector of the fusion protein, host with transformed expression vector, and the application of the fusion protein in medicine for preventing and treating HPV relative diseases. The fusion protein may be expressed in colibacillus in the form of inclusion body and further column renatured and chromatographically purified to reach purity over 95 %. It is used in inducing E6 and E7 specifying cellular immunity reaction in mouse body in the condition of no adjuvant and has obvious therapeutic effect on transplanted TC-1 cell tumor of mouse.

Description

technical field [0001] The present invention relates to a fusion protein of heat shock protein and HPV16Z E6 / E7, a method for obtaining the fusion protein, an expression vector expressing the fusion protein and a host transformed with the expression plasmid, and the fusion protein is effective in treating and preventing HPV-related diseases application in medicines. technical background [0002] HPV is closely related to various epithelial tumors and hyperplasia, and the infection of high-risk HPV is the main cause of female cervical cancer. The detection rate of HPV infection in women's cervical cancer tissues in my country is greater than 99%, among which HPV16 accounts for more than 90%. Worldwide, there are 400,000 new cases of cervical cancer every year, and 200,000 people die from the disease, ranking second in the number of female cancer deaths. At present, the treatment of cervical cancer is mainly surgery and radiotherapy, the 5-year survival rate of which is only...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63A61K39/12A61P35/00
Inventor 王小兵张伟
Owner 张伟
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