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Recombinant protein and medicine composition and application thereof

A recombinant protein and composition technology, applied in the field of biomedicine, can solve the problems of low immunogenicity and inability to induce strong cellular immunity, and achieve strong immunogenicity, good application prospects, and the effect of treating cervical cancer

Active Publication Date: 2017-05-10
南京益康生物医药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of this, the object of the present invention is to provide a recombinant protein and a pharmaceutical composition for application to effectively solve technical defects such as low immunogenicity of recombinant protein vaccines, inability to induce strong cellular immunity, and safety issues in the current technology.

Method used

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  • Recombinant protein and medicine composition and application thereof
  • Recombinant protein and medicine composition and application thereof
  • Recombinant protein and medicine composition and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Embodiment 1: DNA structure and plasmid construction

[0107] E6 / E7 protein of human papillomavirus (HPV) 16 / 18 type is the main oncogenic protein with transforming activity. point mutation.

[0108] In HPV16 type E6, the 47th phenylalanine (F) is mutated to arginine (R), the 50th leucine (L) is mutated to glycine (G), and the 63rd cysteine ​​( C) mutation to glycine (G), cysteine ​​(C) at position 106 to arginine (R); tyrosine (Y) at position 23 in HPV16 E7 was mutated to glycine (G), Cysteine ​​(C) at position 24 is mutated to glycine (G), tyrosine (Y) at position 25 is mutated to glycine (G), cysteine ​​(C) at position 58 is mutated to glycine (G) ), the cysteine ​​(C) at position 91 is mutated to glycine (G).

[0109] In HPV18 type E6, the 49th phenylalanine (F) is mutated to arginine (R), the 52nd leucine (L) is mutated to glycine (G), and the 65th cysteine ​​( C) mutation to glycine (G), cysteine ​​(C) at position 108 to glycine (G); leucine (L) at position 26...

Embodiment 2

[0114] Example 2: Expression and preparation of recombinant protein in Escherichia coli

[0115] The pET28a-Flt3l-RM16 recombinant expression vector was transformed into BL21 (DE3) Escherichia coli competent, coated with kana resistance plate, and the Escherichia coli expressing the recombinant protein was obtained by colony PCR identification. The engineered bacteria (Escherichia coli successfully expressing Flt3l-RM16 recombinant protein) were expanded in LB medium at 37°C, and 0.4mM IPTG was added when OD600=0.6-0.8, and induced at 16°C for 16h. After the induction was completed, the cells were collected by centrifugation at 7500 g for 5 minutes, and the cells were washed twice with PBS (pH 7.4). Take 20g of engineered bacteria and resuspend them in 100ml PBS (PH7.4) and add PMSF (Beyotime) and EDTA (Sinopharm Group) with a final concentration of 1mM, break the bacteria once with a high pressure homogenizer at 1000bar, collect the broken bacteria solution and centrifuge at ...

Embodiment 3

[0119] Embodiment 3: ELISPOT detection cellular immunity level

[0120] Since rm16E6E7 and rm18E6E7; Flt3l-RM16, NCRT-RM16 are similar in structure and function to Flt3l-RM18, NCRT-RM18, therefore, the following Examples 3-6 use rm16E6E7, Flt3l-RM16, NCRT-RM16 as examples.

[0121] Preparation of CpG-ODN: The sequence of CpG-ODN used is shown in Table 1. Utilize solid-phase phosphoramidite triester method chemical synthesis to prepare CpG-ODN, start from 3' end, 1) deprotection group: first use trichloroacetic acid to remove the protection group DMT (two methoxytrityl) to obtain a free 5' hydroxyl group for the next condensation reaction; 2) activation: the phosphoramidite-protected nucleoside monomer is mixed with tetrazolium activator and enters the synthesis column , forming the active intermediate of phosphoramidite tetrazole, which undergoes a condensation reaction with the nucleotide of the deprotected group on CpG; 3) connection: the active intermediate of phosphoramid...

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Abstract

The invention relates to the field of biological medicine, in particular to recombinant protein, a medicine composition and application thereof. The recombinant protein comprises human papilloma virus E6 and E7 plasmodium fusion polypeptides, has specific amino acid sequences, and special space structures, so that strong immunogenicity, particularly cell-mediated immunity can be realized; the human body safety problem is solved through point mutation. The medicine composition provided by the invention comprises the recombinant protein and auxiliary agents, and can stimulate and reinforce the specific T-cell immune response aiming at the human papilloma virus E6 and E7 protein, can be used for effectively treating cervical cancer, and has good application prospects.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a recombinant protein, pharmaceutical composition and application. Background technique [0002] Cervical cancer is a malignant tumor that seriously endangers women's health, and its incidence rate ranks second among female malignant tumors, second only to breast cancer. About 2.645 billion women (over 15 years old) in the world are at risk of cervical cancer. There are 530,000 new cases of cervical cancer every year, accounting for 12% of all female tumors, and 265,000 deaths. About 552 million women over the age of 15 in China are at risk of developing cervical cancer. According to the latest statistics, 61,691 women in China are diagnosed with cervical cancer every year, of which 29,526 die. Only 3.8% of the cases are infected with HPV16 / 18 In a certain period of time, it showed normal cytological features, and 76.1% developed into invasive cervical cancer. [0003] It is known th...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K39/12A61P31/20A61P35/00
CPCA61K39/12C07K14/005A61K2039/585A61K2039/575C07K2319/00C12N2710/20034C12N2710/20022C07K14/4728C07K14/52C12N15/70A61K38/1738A61K38/19A61K39/39A61P31/20A61P35/00C12N2800/22A61K2039/892A61K2039/572Y02A50/30
Inventor 李建强葛君陈思陆凤任苏林周童戚凤春赵志强刘俊林殷相平蒲秀英徐继英王建锋焦文强
Owner 南京益康生物医药有限公司
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