A kind of respiratory syncytial virus f2 protein subunit vaccine and preparation method thereof

A subunit vaccine and syncytial virus technology, applied in the field of respiratory syncytial virus F2 protein subunit vaccine and preparation thereof, can solve the problems of relying on cell culture to obtain, unfavorable for vaccine popularization, and high environmental requirements, and achieves easy culture and fermentation, Simple structure and low production cost

Inactive Publication Date: 2011-12-28
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, currently based on F protein-related vaccines, it is difficult to obtain expression in prokaryotic expression systems, and it is completely dependent on cell culture to obtain
It has high environmental requirements and is expensive, which is not conducive to the popularization of vaccines

Method used

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  • A kind of respiratory syncytial virus f2 protein subunit vaccine and preparation method thereof
  • A kind of respiratory syncytial virus f2 protein subunit vaccine and preparation method thereof
  • A kind of respiratory syncytial virus f2 protein subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Obtaining of F2 gene

[0026] Amplify the F / 18T / DH5α strain (preserved in our laboratory, containing the full gene of RSV fusion protein F), and then use the plasmid extraction kit from Biotech to extract the plasmid (the following plasmids are extracted according to the instructions), so as to The plasmid was used as a template, and the F2 gene was amplified with TIANGEN’s Taq DNA polymerase, and the Nco I, Bam HI two restriction sites and 6×His tag, the primer sequence used is: F2PFNP1: 5'-CCGCCATGGAGGCTTCCAGTCAAAAACATCACTGAAGAATTT-3', F2PFNP2: 5'-CGCGGATCCTTAGTGGTGGTGGTGGTGGTGCCTTCTGGCTCGATTGTTGGCTG-3'; PCR amplification conditions: 94°C 5min; 94 ℃ for 30s, 50℃ for 30s, 72℃ for 30s (30 cycles); 72℃ for 5min; the amplified product was recovered and identified by 1% agarose gel electrophoresis, and the F2 gene with a size of 290bp was amplified (see figure 1 ).

Embodiment 2

[0027] Example 2 Recombinant expression vector F2 / pFN18K and Recombinant engineering bacteria F2 / pFN18K / KRX Construct

[0028] The F2 gene was purified using the Gel Recovery Kit from Biotech (the following gel recovery and purification were performed according to the instruction manual) and the pFN18K plasmid was extracted using the small amount of plasmid extraction kit from Biotech (purchased from Promega, USA). Nco I and Bam H Ⅰ (purchased from TAKARA company) at 37°C for double digestion of F2 gene and pFN18K plasmid for 3 hours. Recover the double-digested F2 fragment and pFN18K fragment with a gel recovery kit. Take 180ng of F2 fragment and 90ng of pFN18K fragment, use 0.5μl T4 DNA ligase (from TAKARA company, CK5021B) to connect overnight at 16°C, and transform the ligation product by heat shock method with CaCl 2 Prepared E. coli KRX Competent Cells (purchased from Promega, USA), coated with Kan + -LB plate, cultivated at 37°C for 16 hours, picked several s...

Embodiment 3

[0029] Example 3 Induced expression of Halo-F2 protein

[0030] Pick a single colony of recombinant engineering bacteria F2 / pFN18K / KRX and inoculate it into Kan + -In LB culture medium, cultivate overnight at 37°C with shaking, and inoculate fresh Kan at 1% the next day + -In LB culture medium, shake culture at 37°C until OD600 is about 0.8, take appropriate samples and add rhamnose (sourced from Sanland-chem International Inc, 102189) at a final concentration of 1 μl / ml respectively, and induce expression at 28°C Overnight, after the end of the induction, take an appropriate amount of samples for SDS-PAGE to determine whether the size is correct. The apparent molecular weight of the Halo-F2 protein is 13.3KDa (see image 3 ), which is basically consistent with the theoretical calculation value, expressed in two forms of inclusion body and soluble protein.

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Abstract

The invention discloses a respiratory syncytial virus F2 protein subunit vaccine and a preparation method thereof. The method constructs a recombinant expression vector F2/pFN18K, and transforms the recombinant expression vector into Escherichia coli to form a recombinant engineering bacterium. The recombinant engineering bacterium undergoes Fermentation culture, high-efficiency expression of Halo-F2 protein was obtained in Escherichia coli by rhamnose induction, and F2 protein with high purity was obtained by digestion with tobacco plaque virus protease (TEV) and chromatographic purification; animal experiments of the present invention showed that, F2 protein has strong immunogenicity and can stimulate the body to produce a better immune response. It is used for human or animal vaccination to resist respiratory syncytial virus infection. Prokaryotic expression of F2 protein in Escherichia coli can improve the expression level of protein vaccines , which has important practical significance for large-scale production and clinical application.

Description

technical field [0001] The invention relates to a respiratory syncytial virus F2 protein subunit vaccine and a preparation method thereof, belonging to the field of biotechnology. Background technique [0002] Respiratory syncytial virus (RSV) is one of the main pathogens that cause bronchiolitis, pneumonia, and bronchitis in infants and young children, and can lead to death in infants and young children. Immunodeficient patients and the elderly are also susceptible to RSV. Since the discovery of the virus, although some progress has been made in RSV research, there are currently only two monoclonal antibodies approved for the treatment of severely ill patients. An effective, completely safe RSV vaccine has yet to emerge. The World Health Organization (WHO) has listed RSV vaccine as one of the priority vaccines in the global vaccine development plan. [0003] However, although the inactivated RSV vaccine used in the 1960s could induce the production of neutralizing antibo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/155A61P31/14C12N15/70C12R1/19
CPCY02A50/30
Inventor 井申荣杨靖佳曾韦锟黄芬
Owner KUNMING UNIV OF SCI & TECH
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