Fusion gene and application in preparation of pneumococcal vaccines

A fusion gene and gene technology, applied in the field of medicine and biology, can solve the problems of poor immunogenicity and no retention of pathogenic particles, etc., achieve high expression, easy purification and preparation, and enhance the effect of immunogenicity

Active Publication Date: 2018-03-23
嘉兴迈维代谢生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the subunit vaccines developed for P1 and P30 proteins are relatively safe, they do not retain a relatively complete pathogenic particle, and generally have poor immunogenicity

Method used

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  • Fusion gene and application in preparation of pneumococcal vaccines
  • Fusion gene and application in preparation of pneumococcal vaccines
  • Fusion gene and application in preparation of pneumococcal vaccines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Construction of pET30a-P1a-Linker-P30a fusion gene

[0026] The P1a-linker-P30 sequence uses the Mycoplasma pneumoniae M129 strain as the sequence template. P1a and P30a respectively select the 3478-4047 bases of the M129 strain P1 and the 311-792 bases of the P30, and connect them through Linker. The company synthesized it on the PUC19-P1a-Linker-P30a vector, cut it with NcoI and XhoI, and connected it to the pET30a vector.

[0027] The PUC19-P1a-Linker-P30a enzyme digestion system is as follows:

[0028]

[0029] The enzyme digestion system of pET30a vector is as follows:

[0030]

[0031] The pUC19-P1a-Linker-P30a enzyme digestion system and the pET30a vector enzyme digestion system were reacted at 37°C for 2 hours, the digestion results were detected by 1% agarose gel electrophoresis, and the target fragment was purified and recovered. Ligate the vector and target fragments with T4 ligase,

[0032] Its reaction system is as follows:

[0033] ca...

Embodiment example 2

[0044] Example 2: Prokaryotic expression of recombinant plasmid pET30a-P1a-Linker-P30a

[0045] Plasmid Transformation The expression plasmid pET30a-P1a-Linker-P30a (Kana-resistant) of the P1a-P30a fusion gene was transformed into Escherichia coli BL21 competent cells.

[0046] The specific operation is as follows:

[0047] 1) Transfer 50 μL of Escherichia coli BL21 competent cell suspension to a sterile 1.5ml EP tube, add 1 μL of the ligation product, swirl gently to mix the contents, and place on ice for 30 minutes.

[0048] 2) Heat shock in a water bath at 42°C for 90s (let the plasmid enter competent cells).

[0049] 3) Take it out and place it on ice for 5 minutes (let the competent cells close).

[0050] 4) Add 200 μL LB medium to each tube. Warm the medium to 37°C with a water bath, then transfer the centrifuge tube to a shaker at 37°C, and incubate for 60 minutes to recover the bacteria. In order to achieve effective conversion, the rotating speed is 180r / min.

[...

Embodiment example 3

[0053] Example 3: Prokaryotic expression of recombinant plasmid pET30a-PLY-Linker-P1a-Linker-P30a

[0054] Plasmid Transformation The expression plasmid pET30a-PLY-Linker-P1a-Linker-P30a (Kana-resistant) of the PLY-P1a-P30a fusion gene was transformed into Escherichia coli BL21 competent cells.

[0055] The specific operation is as follows:

[0056] 1) Transfer 50 μL of Escherichia coli BL21 competent cell suspension to a sterile 1.5ml EP tube, add 1 μL of the ligation product, swirl gently to mix the contents, and place on ice for 30 minutes.

[0057] 2) Heat shock in a water bath at 42°C for 90s (let the plasmid enter competent cells).

[0058] 3) Take it out and place it on ice for 5 minutes (let the competent cells close).

[0059] 4) Add 200 μL LB medium to each tube. Warm the medium to 37°C with a water bath, then transfer the centrifuge tube to a shaker at 37°C, and incubate for 60 minutes to recover the bacteria. In order to achieve effective conversion, the rotati...

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Abstract

The invention relates to a fusion gene. The fusion gene is characterized in that a dominant antigen area P1a-P30a of mycoplasma pneumoniae P1 and P30 is subject to fusion expression; the PLY-P1a-P30ais subject to fusion expression by a gene of pneumolysin PLY and one part of genes of mycoplasma pneumoniae P1 and P30; the immunogenicity of the protein PLY-P1a-P30a is obviously better than the immunogenicity of P1a-P30a; the immunogenicity of the mycoplasma pneumoniae subunit P1a-P30a is enhanced; the immunity function of streptococcus pneumoniae is realized, and the toxicity of mouse is not observed in clinical application; the fusion gene PLY-P1a-P30a is a gene resource with lower cost, the expression quantity of subunit vaccine of the fusion gene PLY-P1a-P30a is high, and the purification and preparation are easy; the stability and immune activity can be maintained for two years or more at the temperature of -80 DEG C; compared with the conventional deactivating vaccine and weak-toxic vaccine, the preparation cost is obviously reduced, and the foundation is laid for the study of mycoplasma pneumoniae and streptococcus pneumoniae vaccines.

Description

technical field [0001] The invention relates to the field of medical biotechnology, in particular to a preparation method and application of a fusion gene of Streptococcus pneumoniae and Mycoplasma pneumoniae genes, pneumolysin and its preparation and application as an immune enhancer. Background technique [0002] Pneumonia is an inflammation that occurs in the terminal airways, alveoli, and interstitium of the lungs. It is a common respiratory infectious disease. It is usually caused by the downward spread of inflammation caused by the failure of complete treatment after upper respiratory tract infection or the low immunity of patients. WHO statistics show (GURGEL R Q, BEZERRA P G, DUARTE MDO C, et al. Relative frequency, possible risk factors, viral code detection rates, and seasonality of respiratory syncytial virus among children with lower respiratory tract infection in Northeastern Brazil[J]. ), 2016, 95(15): e3090.), in recent years, the mortality rate of acute respi...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/66A61K39/116A61K39/09A61K39/02A61P31/04
CPCA61K39/0241A61K39/092A61K2039/53A61K2039/70C07K14/30C07K14/3156C07K2319/00C12N15/66
Inventor 李晓菊文良均
Owner 嘉兴迈维代谢生物科技有限公司
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