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Streptococcus pneumoniae vaccine and preparation method thereof

A Streptococcus pneumoniae and vaccine technology, applied in peptide preparation methods, botany equipment and methods, biochemical equipment and methods, etc., can solve the problems of limited serotypes, protective antibodies produced by infants and young children, and achieve the goal of disease prevention Effect

Active Publication Date: 2020-03-24
遵义医科大学珠海校区
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PPV23 is currently the only polysaccharide vaccine used to prevent Streptococcus pneumoniae infection in children over 2 years old and high-risk groups, but PPV23 cannot induce protective antibodies in infants under 2 years old; while vaccination with PCV13 can be used to prevent Streptococcus pneumoniae in children under 5 years old Streptococcus pneumoniae-associated disease, but coverage of serotypes is limited

Method used

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  • Streptococcus pneumoniae vaccine and preparation method thereof
  • Streptococcus pneumoniae vaccine and preparation method thereof
  • Streptococcus pneumoniae vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] A Streptococcus pneumoniae antigen SPD_0151 protein, the amino acid sequence of which is SEQ ID NO.1.

[0031] Its preparation method comprises the following steps:

[0032] (1) Link the spd-0151 gene (its sequence is SEQ ID NO.2) without signal peptide sequence to the pGEX-4T-1 vector to obtain the expression vector pGEX-4T-0151;

[0033] (2) Introduce the expression vector pGEX-4T-0151 into Escherichia coli to obtain the expression strain; inoculate the expression strain in LB medium containing 100 ng / μL ampicillin, culture it with shaking at 37°C overnight, and continue to expand the culture; the strain to be expressed Proliferate until the OD600 in the logarithmic phase of growth is 0.6-0.8, add IPTG (isopropyl-β-D-thiogalactoside) to the culture system to a mass concentration of 0.5mmol / L, induce expression for 6h, and collect the bacteria Body, cleavage to obtain protein;

[0034] (3) GST affinity chromatography is used to purify the obtained protein to obtain t...

Embodiment 2

[0036] Experimental group: the mixture of the target protein SPD_0151 and aluminum adjuvant obtained in Example 1, the concentration of the target protein SPD_0151 in the mixture is 125 μg / mL, the concentration of the aluminum adjuvant is 0.5 mg / mL, and the composition of the aluminum adjuvant used is Aluminum hydroxide;

[0037] Control group: a mixture of 1×PBS and aluminum adjuvant, the concentration of the aluminum adjuvant in the mixture is 0.5 mg / mL, wherein the component of the aluminum adjuvant used is aluminum hydroxide.

[0038] The above-mentioned experimental group and control group were used to subcutaneously inject BALB / c mice at multiple sites, the injection amount was 25 μg / mouse, and 10 mice in each group were immunized three times with an interval of 14 days between each immunization. Seven days after the completion of the third immunization, the antibody titer in the mouse serum was determined by enzyme-linked immunosorbent assay.

[0039] Usually, the immu...

Embodiment 3

[0042] The serum (antibody containing SPD_0151 protein) of the mice immunized by the experimental group in Example 2 was used as the primary antibody, and was detected by Western blot (Western blot).

[0043] Such as image 3 As shown, the antibody in the mouse serum can specifically recognize the SPD_0151 protein, but the mutant of the SPD_0151 protein cannot be detected, and the detection result is a single band, indicating that the antibody has good specificity; and the antibody can not only be used to detect prokaryotic expression Purified SPD_0151 protein, and can detect SPD_0151 protein expressed in Streptococcus pneumoniae, wherein the internal reference is Gap antibody (GAPDH, purchased from Beijing Quanshijin Biotechnology Co., Ltd.).

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Abstract

The invention belongs to the field of biological products, and discloses a streptococcus pneumoniae vaccine, which comprises SPD_0151 protein and an immunologic adjuvant, and the amino acid sequence of the SPD_ 0151 protein is SEQ ID NO.1. The invention also discloses a preparation method of the SPD_0151 protein, which comprises the following steps: inserting spd_0151 gene which contains no signalpeptide sequence into a recombinant plasmids, introducing the recombinant plasmids into expression bacteria, inducing expression, and purifying to obtain the SPD_0151 protein, wherein the sequence ofthe spd_0151 gene is shown as SEQ ID NO.2. The streptococcus pneumoniae vaccine has a good protection effect on challenge of streptococcus pneumoniae strains of different subtypes, and can effectively enhance the resistance to streptococcus pneumoniae.

Description

technical field [0001] The invention belongs to the field of biological products, in particular to a Streptococcus pneumoniae vaccine and a preparation method thereof. Background technique [0002] Streptococcus pneumoniae is a Gram-positive bacterium that often resides in the oral cavity and nasopharynx of normal people. Generally, it does not cause disease and only forms a carrier state. It only causes disease when the immunity is weakened. Streptococcus pneumoniae is one of the most common causes of community-acquired pneumonia, and the bacteria can also cause many other serious diseases, including meningitis, otitis media, bacteremia and sepsis, especially in children, the elderly, and AIDS patients and other immunocompromised patients, posing a major threat to human health worldwide. [0003] Vaccines and antibiotics are currently the main means of preventing and treating Streptococcus pneumoniae infection. Pneumococcal vaccines currently on the market are mainly 23-v...

Claims

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Application Information

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IPC IPC(8): A61K39/09A61P31/04A61P11/00C12N15/31C12N15/70C07K14/315C07K1/22C07K1/18C07K1/16
CPCA61K39/092A61P31/04A61P11/00C07K14/3156C12N15/70A61K2039/70Y02A50/30
Inventor 阳小燕乐尧金
Owner 遵义医科大学珠海校区
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