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Respiratory syncytial virus sub-units vaccine, preparation and application

A subunit vaccine, syncytial virus technology, applied in the field of respiratory syncytial virus vaccine and its preparation, vaccine preparation of avirulent E. To enhance the degree of disease and other issues, to achieve the effect of strengthening local mucosal immune function and systemic immune function, eliminating pathological reactions, and improving expression levels

Inactive Publication Date: 2008-09-17
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the 1960s, when children were immunized with inactivated RSV vaccines, they could induce the production of neutralizing antibodies, but they were not protective. Moreover, the use of vaccines enhanced the incidence of children's subsequent infection with wild strains of RSV, and significantly increased hospitalization and mortality.
The preparation of vaccine antigens in these two documents is produced and purified by whole virus cell culture, the follow-up process is complicated and the production cost is high

Method used

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  • Respiratory syncytial virus sub-units vaccine, preparation and application
  • Respiratory syncytial virus sub-units vaccine, preparation and application
  • Respiratory syncytial virus sub-units vaccine, preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Truncated RSV Membrane Protein G and Mutant G CTL Acquisition of genes:

[0047] Entrusted a DNA synthesis company to synthesize the entire gene sequence of RSV G protein nt288~690, a total of 303 bp, and primers gc1: 5′-CTTTCTTTTTCCAGGTAAGTTGGATTGTTGCTGCAGATGC-3′; gc2: 5′-GAAAGTATTTTATAAAAGAATACCAAACAAAAAACCTG-3′; gp10: 5'-GCGGATCCTTAGTGGTGGTGGTGGTGGTGAGGCTTGGTGGTAGGTACTTC-3'. Among them, gp1 and gp10 are used to amplify the G protein gene, and introduce Nde I and BamH I restriction sites and 6×HIS tags, and gc1 and gc2 are used to mutate the CX3C motif in the G gene and replace it with a CTL epitope. Primers (gp1, gp10) were used for PCR amplification, and the PCR product (about 320bp) was the truncated G gene. G was obtained by overlap extension PCR method CTL Gene, that is, use the G gene synthesized by the whole gene as a template, and use primers gp1 / gc1 and gc2 / gp10 to amplify, respectively, to obtain fragments g11 and g210. amplified to obtain the...

Embodiment 2

[0048] Embodiment 2: recombinant expression vector G / pET22b and GCTL Construction of / pET22b:

[0049] G gene and G CTL After gene purification, digest with Nde I and BamH I at 37°C for 2 hours, recover the purified fragment, and connect with the purified expression vector pET22b double digestion product with T4 DNA ligase at 16°C overnight, and transform the ligated product by heat shock method CaCl 2 Prepared E.coli DH5α, coated with Amp + -LB plate, cultured at 37°C for 16 hours, picked several single colonies to inoculate Amp + -LB culture medium, cultured with shaking at 37°C for 12 hours, extracted the plasmid by alkaline lysis, and identified the G gene and G CTL Gene, and the sequencing result of a professional DNA sequencing company is correct. Construct the correct recombinant plasmid and transform E.coli BL21(DE3) again using the above method to induce the expression of truncated G protein and G CTL protein.

Embodiment 3

[0050] Example 3: G protein and G CTL Induced expression of protein:

[0051] Single colonies of recombinant engineered bacteria G / pET22b / BL21 and GCTL / pET22b / BL21 were picked and inoculated into Amp + -In LB culture medium, cultivate overnight at 37°C with shaking, and inoculate fresh Amp at 1% the next day + -In LB culture medium, shake culture at 37°C until OD600 is about 1.0, then add 0.5mmol IPTG to induce expression at 37°C for 4 hours. After induction, an appropriate amount of samples were taken for identification by SDS-PAGE and Western blot. The results showed that: G protein and G CTL The apparent molecular weights of the proteins are 14.2KDa and 14.7KDa respectively, which are basically consistent with the theoretically calculated values, and the relative contents of the total protein in the bacteria are 18.4% and 19.4% respectively.

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Abstract

The invention relates to a respiratory syncytial virus vaccine, and the preparation method and application, in particular to an application of escherichia coli to express and recombinant respiratory syncytial virus G protein and mutation G protein, and preparation vaccine with nontoxic typed escherichia coli heat labile enterotoxin, for human or animal preventive inoculation, and for respiratory syncytial virus resistance, belonging to the field of biotechnology. The respiratory syncytial virus vaccine is characterized in that: the respiratory syncytial virus subunits vaccine comprises lopped respiratory syncytial virus RSV protein G, and further comprises nontoxic typed escherichia coli heat labile enterotoxin LT adjuvant. The vaccines are lopped respiratory syncytial virus RSV protein G containing amino acid between aa130 and 230 of the original G protein, and substitutes the amino acid CAWIC (CX3C module ordered) between aa182 and 186 with the amino acid YLEKESIYY (CTL epitope) on the RSV M protein, forming the GCIL protein. The respiratory syncytial virus vaccine has the advantages of remarkable practical significance for preventing human respiratory syncytial virus infection.

Description

Technical field: [0001] The invention relates to a respiratory syncytial virus vaccine and a preparation method thereof, more specifically expressing recombinant respiratory syncytial virus G protein and mutant G protein using Escherichia coli, and preparing the vaccine with avirulent E. It is used for human or animal vaccination to resist respiratory syncytial virus infection, and belongs to the field of biotechnology. Background technique: [0002] Human respiratory syncytial virus (Human Respiratory Syncytial virus, RSV) is the most important pathogen of lower respiratory tract infection in infants and young children worldwide. Immunodeficient patients and the elderly are also susceptible to RSV. In the United States alone, it is estimated that 90,000 infants are hospitalized each year due to RSV infection, with a mortality rate of 2% to 5%. my country not only has sporadic RSV infection, but also has several large-scale outbreaks. Among children with respiratory tract ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/155C12N15/45C12N15/70C07K14/135A61P31/00A61P37/04
Inventor 井申荣魏云林林连兵季秀玲李光
Owner KUNMING UNIV OF SCI & TECH
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