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Expression and application of human papilloma viruses type 16 and 18 L1 proteins in inset cells

A technology of L1 protein and insect cells, applied in the fields of application, antiviral agents, viral antigen components, etc., can solve the problems of high cost and difficult to clearly prove the protective effect, and achieve the effect of increasing the expression level

Inactive Publication Date: 2011-09-14
DALIAN ALEPH BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is costly and protection against less common HPV subtypes is difficult to clearly demonstrate

Method used

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  • Expression and application of human papilloma viruses type 16 and 18 L1 proteins in inset cells
  • Expression and application of human papilloma viruses type 16 and 18 L1 proteins in inset cells
  • Expression and application of human papilloma viruses type 16 and 18 L1 proteins in inset cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The optimization of the gene sequence of the L1 protein of embodiment 1.HPV16 / 18

[0041] The amino acid sequences of the L1 proteins of the highly pathogenic HPV16 and HPV18 strains prevalent in my country are shown in SEQ ID NO.7 and 8, respectively.

[0042] Using the commercially available software OptimumGene TM The L1 gene sequence of HPV16 and HPV18 was optimized to increase the expression level of L1 gene in insect cells. Wherein, the optimized HPV16 L1 gene sequence is shown in SEQ ID NO.1; the optimized HPV18 L1 gene sequence is shown in SEQ ID NO.2.

Embodiment 2

[0043] Example 2. Construction of pVL16-L1 and pVL18-L1 transfection plasmids

[0044] Using the primer pair 5'-CTC GAG GAA TTC AGG ATG CAA GTG ACG TTTATT-3' (SEQ ID NO.3) and 5'-A GAG CTC CCA TGG AGG TTA CAA CTTGCG TTT CTT-3' (SEQ ID NO.4 ), and the primer pair 5′-CTC GAG GAA TTCAGG ATG TGC TTG TAC ACG CGC-3′ (SEQ ID NO.5) and 5′-A GAG CTCCCA TGG AGG TTA TTT GCG AGC TCT CAC G-3′ (SEQ ID NO.5) NO.6), respectively amplifying artificially synthesized optimized L1 genes of HPV16 and HPV18 by PCR. The PCR reaction conditions were pre-denaturation at 95°C for 5 minutes; 30 cycles of denaturation at 95°C for 30 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 2 minutes; finally, extension at 72°C for 10 minutes. After PCR amplification, the L1 gene fragments with Xho I and Nco I restriction endonuclease sites at both ends were obtained.

[0045] Use commercially available Xho I and Nco I (for example, purchased from Bao Biological Engineering (Dalian) Co., Lt...

Embodiment 3

[0047] Example 3. Preparation of recombinant baculovirus

[0048] The pVL16-L1 and pVL18-L1 recombinant plasmids were mixed with AcNPV genomic DNA (from the kit of BD Company of the United States, BD BaculoGold TM , 560129) together to co-transfect Sf-9 cells (purchased from Invitrogen, USA). After cultured at 27°C for 4 days, the volume of transfected cells was observed to increase under the microscope. After culturing for 5 days, the cell culture supernatants (containing recombinant viruses rBV16 and rBV18, respectively) were collected. Cell supernatants were inoculated into freshly cultured Sf-9 cells, and cultured at 27°C for 3 days. Take the culture supernatant and subculture again. Afterwards, take the culture supernatant, add an equal volume of 2×precipitation solution (containing 20% ​​PEG6000, 1M NaCl), and mix overnight at 4°C. The mixture was then centrifuged at 13000 rpm for 30 minutes at 4°C, and the supernatant was discarded. The resulting pellet was resus...

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Abstract

The invention provides a method for preparing a bivalent vaccine for human papilloma viruses HPV16 and HPV18, which comprises the following steps of: optimizing gene sequences of L1 proteins of the HPV16 and HPV18; cloning the optimized gene sequences into a baculovirus expression vector and transfecting the inset cells to obtain a recombinant baculovirus; infecting the insect cells by using the baculovirus; and recovering and purifying the L1 proteins expressing the HPV16 and HPV18, and mixing with an adjuvant to prepare the bivalent vaccine. The invention also provides the optimized gene sequences of the L1 proteins of the HPV16 and HPV18, vectors containing the gene sequences, and cells containing the vectors.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the expression of human papillomavirus type 16 and type 18 L1 proteins in insect cells and a vaccine preparation method thereof. Background technique [0002] Statistics from the World Health Organization (WHO) show that cervical cancer is an invasive disease that seriously endangers women's health. Its incidence rate is second only to breast cancer, ranking second among female malignant tumors in the world, and even in some developing countries. first place. Cervical cancer is one of the most common gynecological malignancies. About 500,000 women around the world suffer from cervical cancer and nearly 300,000 women die from cervical cancer every year. Among them, 80% of the deaths occur in developing countries. According to incomplete statistics, there are about 138,000 cervical cancer patients in my country, and about 50,000 people die of cervical cancer every year, and the incidenc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12C12N15/37C12N15/63C12N15/866C12N5/10C07K14/025A61P31/20
Inventor 王敏文王玉峰周长民闫昆明李春明张千王飞宇聂飞周蕾郭文军高伟星段广宇李卫东
Owner DALIAN ALEPH BIOMEDICAL
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