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Bacterial polysaccharide-protein conjugate vaccine and preparation method thereof

A protein-conjugated vaccine, bacterial polysaccharide technology, applied in the field of conjugated vaccines, can solve problems such as lack of protection

Active Publication Date: 2011-02-16
普大生物科技(泰州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] In 1988, when clinical trials began with another rotavirus strain, the WC3 porcine strain (G6P[5] type), initial results proved to be effective, but no significant protection was shown in subsequent trials, The vaccine has also stopped continuing trials

Method used

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  • Bacterial polysaccharide-protein conjugate vaccine and preparation method thereof
  • Bacterial polysaccharide-protein conjugate vaccine and preparation method thereof
  • Bacterial polysaccharide-protein conjugate vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1 Establishment of cDNA library of rotavirus

[0128] Human rotavirus Wa strain (2018-VR, ATCC) was amplified at 36°C for 1 to 2 days in MA104 cells (monkey embryonic kidney cells) supplemented with 1 μg / ml trypsin. Cell debris was removed by low-speed centrifugation, and hydroxyapatite (HA) was added to the supernatant to purify viral RNA. The specific steps are as follows: 600 μl of 6M guanidine isothiocyanate (GITC) solution is added to 400 μl of culture supernatant, and mixed. 50 [mu]l (two drops) of HA suspension was then added and the tube was mixed by inversion upside down or on a shaker for 20 minutes at room temperature to allow adsorption of nucleic acids to HA. Centrifuge at 13,000 g for 1 min and discard the supernatant. Wash RNA-HA twice with 1 ml of 10 mM potassium phosphate pH 6.8. Add 40 μl of 200 mM potassium phosphate pH6.8 solution to elute RNA from HA crystals, and collect purified RNA by centrifugation at 13000 g for 3 minutes. Perform r...

Embodiment 2

[0134] Example 2 Construction of pET28aWaVP8 expression full-length VP8 plasmid

[0135] The pGEM-TWaVP8 prepared in Example 1 was digested with restriction endonucleases NdeI and BamHI to obtain the insertion sequence of the VP8 gene. The vector pET28a(+) was also digested with restriction endonucleases NdeI and BamHI. Add 10 μg of VP8 gene insertion sequence, T4 DNA ligase and cut pET28a(+) to the test tube, and react overnight at 16°C. The constructed plasmid was called pET28aWaVP8. The protein expressed by the plasmid contains a 6-histidine tag (hexa-histidine tag, 6×His-Tag), and a thrombin recognition site (thrombin recognition site) at the 5' end.

[0136] The pET28aWaVP8 plasmid was transformed into the competent cell E.coli TOP10, and the clone was screened for pET28aWaVP8-positive colonies in an LB culture dish with 50 μg / ml of Kanamycin. Inoculate in LB culture medium for amplification, save the expression strain below -20°C, and extract the pET28aWaVP8 plasmid f...

Embodiment 3

[0137] Embodiment 3 expression recombinant VP8 protein

[0138] 1. Transform the pET28aWaVP8 plasmid prepared in Example 2 into BL21(DE3) competent cells, inoculate the cells into 50 μg / ml kanamycin LB culture dishes, and inoculate at 37°C in CO 2 overnight in the incubator.

[0139] 2. Pick out a colony, inoculate it into 10 milliliters of kanamycin LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.5) containing 50 μg / ml for amplification, and culture at 37°C overnight.

[0140] 3. Transfer the culture solution to 100 ml of 50 μg / ml kanamycin LB culture solution and continue to cultivate. When the OD of absorbance at 600nm reaches 1.0, inoculate the culture solution into 6 liters to 50μg / ml kanamycin LB culture solution, and continue culturing in a shaker at 37°C with a shaking speed of 200rpm until the OD of absorbance at 600nm When it reaches 0.6-0.8, add 0.3mM IPTG (isopropyl-β-D-thiogalactoside) to induce the expression of VP8.

[0141] 4. Under the same cultu...

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Abstract

The invention relates to a bacterial polysaccharide-protein conjugate vaccine with immunogenicity, in particular to a conjugate vaccine which is formed by connecting a recombinant rotavirus protein with a bacterial polysaccharide by using a covalent bond, a nucleotide sequence for coding the recombinant rotavirus protein, a recombinant expression system, a protein expressed by the recombinant expression system, a preparation method of the conjugate vaccine and a pneumococcus polysaccharide-recombinant rotavirus protein conjugate vaccine. The bacterial polysaccharide is connected with a recombinant rotavirus surface protein through a covalent bond. The recombinant rotavirus protein is selected from a partial or complete amino acid sequence of a P-gene rotavirus protein and a partial or complete amino acid sequence of a G-gene rotavirus protein.

Description

technical field [0001] The invention relates to an immunogenic bacterial polysaccharide-protein conjugated vaccine, in particular to a conjugated vaccine which uses rotavirus protein and bacterial polysaccharide to be covalently bonded. Background technique [0002] Preventing diseases through vaccination is an effective method that has been proved to be effective in human clinical practice for more than a century. After years of hard work, the medical community has developed various vaccines to prevent various diseases caused by infection, such as bacteria, viruses and fungi, which has greatly improved human health. The continuous development of biotechnology has promoted the diversification of vaccine varieties. Today, there are vaccines developed by inactivated virus technology to prevent infectious diseases caused by viruses, such as Japanese encephalitis vaccine, polio vaccine, influenza vaccine, etc.; live attenuated vaccines developed by attenuated virus technology, ...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/46A61K39/108C12N15/81A61K39/102C12N15/70A61P31/04A61K39/295A61K39/39A61K39/09A61K39/112A61P31/14C12N15/866A61K39/15A61K39/095A61K39/106C07K14/14
CPCY02A50/30
Inventor 李建平
Owner 普大生物科技(泰州)有限公司
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