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Vector for treatment vaccine for stable and constitutive high-expression cervical cancer and recombinant lactobacillus transformed by same

A vector and vaccine technology, which can be used in the introduction of foreign genetic material, vector, and hybrid cell preparation using vectors, which can solve the problems of limited plant system, low protein expression and purification, etc.

Active Publication Date: 2012-02-01
BIOLEADERS CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the commercial application of this plant system is limited by the low expression of HPV L1 protein and problems related to purification

Method used

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  • Vector for treatment vaccine for stable and constitutive high-expression cervical cancer and recombinant lactobacillus transformed by same
  • Vector for treatment vaccine for stable and constitutive high-expression cervical cancer and recombinant lactobacillus transformed by same
  • Vector for treatment vaccine for stable and constitutive high-expression cervical cancer and recombinant lactobacillus transformed by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Preparation of a surface expression vector (pKV-Pald-PgsAL-amylase) containing a repE mutant gene

[0051] In this example, in order to stably express the vector in host cells, a surface expression vector containing a mutant repE gene and showing an increased expression level of the target protein was constructed.

[0052] First, in order to further increase the expression of the target protein in lactic acid bacteria, a fragment of the aldolase promoter from Lactobacillus casei was obtained. The aldolase promoter was obtained by PCR using pDT-PgsA-amylase (disclosed in Korean Patent Publication No. 10-2008-0086161) as a template and SEQ ID NO: 2 and SEQ ID NO: 3 as primers.

[0053] SEQ ID NO: 2: 5'-cgc gca tgc aat acc cac tta ttg cg-3'

[0054] SEQ ID NO: 3: 5'-cag ttc ttt ttt cat gta gat atc ctc c-3'

[0055] As a result, a DNA fragment of 421bp was obtained, which contained the aldolase promoter, the Sph I restriction endonuclease site and at th...

Embodiment 2

[0062] Example 2: Preparation of HPV16 E7 surface expression vector (pKV-Pald-PgsA-E7)

[0063]Using the surface expression vector (pKV-Pald-PgsA-amylase) prepared in Example 1, the gene encoding the HPV16 E7 protein was inserted into the C-terminal end of PgsA, thereby preparing pKV capable of expressing the target protein on the surface of lactic acid bacteria - Pald-PgsA-E7 vector.

[0064] For this reason, remove the pKV-Pald-PgsA-amylase vector prepared in Example 1 with pGS The fused amylase gene, and the HPV16 E7 coding gene was inserted into the vector. like figure 1 As indicated, using pHAT:PgsA-E7 (Poo et al., Int. J. Cancer, 119:1702, 2006) as a template and SEQ ID NO: 6 and SEQ ID NO: 7 as primers to obtain a fragment containing the HPV16 E7 gene by PCR.

[0065] SEQ ID NO: 6: 5'-gcg gga tcc cat gga gat aca cct aca ttg c-3'

[0066] SEQ ID NO: 7: 5'-acg cag aag cgg tct gat aa -3'

[0067] As a result, a 386bp fragment containing the HPV16 E7 gene was...

Embodiment 3

[0070] Embodiment 3: Check the expression of pHAT:PgsA-E7 and pKV-Pald-PgsA-E7 in transformed lactic acid bacteria

[0071] In this example, the pHAT constructed in Example 2: PgsA-E7 and pKV-Pald-PgsA-E7 were respectively used to transform Lactobacillus casei. The transformed recombinant Lactobacillus casei strain was cultured, and the expression of E7 protein in the recombinant strain was examined. Expression of E7 protein fused to PgsA in transformed recombinant L. casei strains was examined.

[0072] Recombinant Lactobacillus casei strains transformed with pHAT: PgsAE7 and pKV-Pald-PgsA-E7 were cultured statically in MRS medium (Lactobacillus MRS, Becton Dickinson and Company Sparks, USA) at 30°C to induce the interaction with poly-γ - glutamate synthase pGS Surface expression of HPV16 E7 protein fused to the C-terminus of the gene.

[0073] Whole cells of the cultured Lactobacillus casei strain were subjected to SDS polyacrylamide gel electrophoresis and Western...

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Abstract

The present invention relates to a surface expression vector for HPV vaccine preparation including a gene that encodes an antigen protein related to the occurrence of human papilloma virus tumors, connected with: a composite gene with repE mutation gene having the amino sequence of sequence No. 1, a promoter, a poly-gamma-glutamate synthetase complex, and a poly-gamma-glutamate synthetase complex gene. The recombinant lactobacillus of the present invention that has been transformed into a surface expression vector of the antigen protein of the human papilloma virus, and the composition using the same, may be utilized as a treatment vaccine for cervical cancer and directly applied to the cervical or vaginal area, so that it has very economical effects.

Description

Technical field [0001] The present invention involves the surface expression carrier used to prepare HPV therapeutic vaccines, where the surface expression carrier contains genes with an amino acid sequence SEQ ID NO: 1: Repert mutant protein, promoter, poly-γ-glutamic acid synthetic enzymeThe genes connected to the composite-γ-glutamic acid synthetase composite gene, which encodle the tumor-induced antigen protein related to the human papilloma virus. technical background [0002] It is known that the use of exogenous proteins for expressing a large amount of exogenous protein depends on the number of copies of genes, the number of plasmids, and a promoter strength for controlling transcription.According to reports, in addition to the highly used high -expression promoter, it can increase the expression of target protein by changing the number of single bacterial internal quality particle carriers (Tomio, M., etc., etc. Appl. Microbiol. Biotechnol ., 28: 170, 1988).Similarly, it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12N15/03C12N15/37A61K39/12
CPCC12N2810/55C12N15/746C12N2710/20045A61K39/12C12N2710/20034A61K2039/542A61K2039/523A61P31/20A61P35/00A61P37/04A61K39/0011C07K14/025C07K14/335C12N15/69C12N15/03C12N15/11C12N15/86
Inventor 成文喜夫厦玲李日汉
Owner BIOLEADERS CORP
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