CN1248631A

CN1248631A - Fusion protein for immunoprophyaxis and immunotherapy of venereal disease and cancer

Info

Publication number: CN1248631A
Application number: CN 98112264
Authority: CN
Inventors: 周国庆
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-09-24
Filing date: 1998-09-24
Publication date: 2000-03-29

Abstract

The present invention makes the heat shock protein of mycobacterium bovis var. BCG (M. Bovis BCG) fuse on tumor specific antigen, i. e. human papillomavirus (HPV) to form fusion protein. This fusion protein can be expressed in colibacillus, microzyme and plant, and (this fusion protein) is used to make immune injection, it can produce specific cell immune and humoral immune reaction of tumor specific antigen, not only possesses the immunoprophylaxis capacity for preventing human papillomavirus (HPV) from infection, but also can be used for immunoprophylaxis and immunotherapy of pointed condyloma, tumor and cancer caused by HPV, so that said invented recombinant fusion protein provides a simple and effective prophylactic immune preparation and therapeutic immune preparation.

Description

The fusion rotein of a kind of immunoprophylaxis and immunotherapy of venereal disease and cancer

The invention belongs to biotechnology---the genetically engineered field.It is characterized in that: the albumen (as early protein E4 or E5 or the E6 or the E7 of the 16th type (HPV-16)) that merges one section or whole human papillomavirus's (HPV) early expression, heat shock protein(HSP) (Heat shock protein, i.e. Hsp at bacterium mycobacterium bovis var.BCG (M.BOVIS BCG).Heat shock protein(HSP) as 65kDa or 70kDa) on, constitutes fusion rotein.The fusion rotein of this type is expressed in intestinal bacteria, yeast or plant.This albumen not only has the ability that immunoprophylaxis HPV infects, and has the ability of pointed condyloma, tumour and cancer that immunoprophylaxis and immunotherapy cause by HPV.

It is the most extensive at present infectious venereal disease that human papillomavirus (HPV) infects.It is general that reproductive organ HPV infects, and take place in age 15-49 year the men and women of the U.S. about 10%, and most of such infection is subclinical (promptly not having clinical manifestation).In PCR detected, HPV-16 almost was present among all woman, and is irrelevant with the cytology result.Wherein 1% HPV-16 type male women will develop into diffustivity uterus carcinoma (in China, above-mentioned data are with much higher).No matter the man, or among the woman, infected at external genital by HPV and produce strumae, claim pointed condyloma.HPV infects also to bring out and produces tumor of cervix and cancer in the women.Serious HPV infects all will influence Fertility, also cause the rugged shape of infant development.

The pointed condyloma that infected by HPV to cause, existing treatment is an integrating laser carbonization in China, or the serious Interferon, rabbit of beating, yet efficient low, only controls table and does not control root, generally some months will recur.Because be virus infection, there is not specifics to control.

Cervical cancer is women's the most general second largest malignant diseases, is only second to mammary cancer.But,, all also only limit to excision of operation property and radiation treatment no matter be to infection disease or to tumor treatment between the more common cervical epithelial cell of having diagnosed out.

Therefore, in the therapeutic immunization mode cell that infects HPV is carried out the great potentiality of immunotherapy tool.In addition, to the HPV immunization can success induce secular immune response, thereby proof can prevent HPV to infect chronically, prevents the generation of pointed condyloma and tumour.

The objective of the invention is to, create a kind of fusion rotein, a kind of new simple and effective immunoprophylaxis and the novel method of immunotherapy are provided with gene engineering method.Not only can immunoprophylaxis HPV infect, and can treat pointed condyloma and the cancer that infects by HPV and bring out.

Embodiment

(1) structure of recombination fusion protein

Merging heat shock protein(HSP) (Hsp) goes up and the formation fusion rotein to the antigen (HPV) of tumour-specific.With recombination fusion protein Hsp-E7 is example, and 539 amino acid of its N-end come from the heat shock protein(HSP) of the 65kda (kilodalton) of mycobacteriumbovis var.BCG; Terminal 98 amino acid of C-come from the early protein E7 of human papilloma (HPV) Class1 6, and it is proteinic amino in proper order as Fig. 1, about 67737 dalton of the molecular weight of expectation.

The N-end of HspE7 can connect the leading order of a plurality of Histidines (Histidine), is convenient to purifying.

(2) expression of recombination fusion protein HspE7 in intestinal bacteria.

Recombination fusion protein is cloned on the pet28a carrier, and its promoter is T7, can be by this recombinant protein of IPTG abduction delivering.Recombination fusion protein also can be cloned on other colibacillary carrier, is expressed in the intestinal bacteria.

(3) fusion protein expression is in plant

Recombination fusion protein can be cloned on pBI121 or pBI221, changes in the plant by Agrobacterium and particle gun respectively, expresses in plant.Recombination fusion protein also can be cloned on other plasmid vector, is expressed in the plant.

(4) recombination fusion protein can be cloned on the zymic plasmid vector, expresses in yeast.

Embodiment:

(1) recombination fusion protein Hsp-E7 is cloned on the pet28a carrier, and its promoter is T7, at expression in escherichia coli.

(2) recombination fusion protein can be cloned on pBI121 or pBI221, expresses in plant.Its promoter is 2 * CaMV35S+AMV RNA4 promoter and enhanser.

(3) recombination fusion protein can be cloned on the zymic plasmid vector, expresses in yeast.

(4) with recombination fusion protein Hsp-E7 immunity mouse, not only inducing cell immunity but also induce humoral immunization.These immunity are specific to HPV-16-E7.Further, carry out epidemic prevention, be protected from the challenge of infecting of HPV and tumour with this recombination fusion protein in physiological saline; And curative immunity causes already present tumour to disappear.Although being (Histidine-tagged) recombination fusion protein (h) Hsp-E7 with the leading order of a plurality of Histidines of tool, most data obtains, but directly relatively recombination fusion protein (h) Hsp-E7 is with reorganization but there is not the fusion rotein Hsp-E7 of the leading order of Histidine (Histidine-tag), and the two all has the ability that the equal HPV of prevention infects, eliminates pointed condyloma and tumour.

Claims

1. the present invention is a kind of fusion rotein for the treatment of the cancer that venereal disease and do as one likes disease cause.The method of claim recombination fusion protein, the heat shock protein(HSP) (Heatshock protein) that promptly merges mycobacterium bovis var.BCG is to human papillomavirus's (HPV) viral protein.

2. the method for claim 1, heat shock protein(HSP) a part of all be blended in human papillomavirus's viral protein a part of or all on, constitute fusion rotein.

3. as claim 1,2 described methods, the amino acid of recombination fusion protein N-end comes from the heat shock protein(HSP) of mycobacterium bovis var.BCG, and the C-end amino acid comes from the albumen (as E4 or E5 or E6 or the E7 of HPV-16) of the early expression of human papillomavirus (HPV) Class1 6 (HPV-16) or Class1 8 (HPV-18).

4. as claim 1,2 described methods, the amino acid of recombination fusion protein N-end comes from the albumen (as E4 or E5 or E6 or the E7 of HPV-16) of the early expression of human papillomavirus (HPV) Class1 6 (HPV-16) or Class1 8 (HPV-18), and the C-end amino acid comes from the heat shock protein(HSP) of mycobacterium bovisvar.BCG.

5. as claim 1,2,3 and 4 described fusion roteins, the N-end of recombination fusion protein can connect the leading order (Histidine-tagged) of a plurality of Histidines (Histidine), is convenient to purifying.

6. as claim 1,2,3,4,5 described recombination fusion proteins, can be expressed in intestinal bacteria, yeast and the plant by plasmid vector.

7. as claim 1,2,3 and 5 described recombination fusion proteins, the amino acid of its N-end comes from 65kDa or the 70kDa heat shock protein(HSP) of mycobacterium bovis var.BCG, and the C-end amino acid comes from the albumen (as E4 or E5 or E6 or the E7 of HPV-16) of the early expression of human papillomavirus (HPV) Class1 6 (HPV-16) or Class1 8 (HPV-18).

8. recombination fusion protein Hsp-E7 as claimed in claim 7, the amino acid that its N-is terminal 539 comes from the 65kDa heat shock protein(HSP) of mycobacterium bovis var.BCG, and terminal 98 amino acid of C-come from the albumen E7 of the early expression of human papillomavirus (HPV) Class1 6 (HPV-16).

9. recombination fusion protein Hsp-E7 as claimed in claim 8, its amino-acid sequence such as Figure of description 1.

10. as claim 8,9 described recombination fusion protein Hsp-E7, its N-end can connect the leading order of a plurality of Histidines (Histidine), is convenient to purifying.

11. can be with recombination fusion protein (h) Hsp-E7 of a plurality of Histidines (Histidine) as claim 8,9,10 described recombination fusion protein Hsp-E7 and N-end, not only have the ability that immunoprophylaxis human papillomavirus (HPV) infects, and have the effect of pointed condyloma, tumour and cancer that immunoprophylaxis and immunotherapy cause by human papillomavirus (HPV).