Methicillin-resistant staphylococcus aureus (MRSA) recombinant multivalent subunit genetic engineering vaccine and method for preparing same

A genetically engineered vaccine, methicillin-resistant technology, applied in the field of biopharmaceuticals

Active Publication Date: 2010-09-29
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SEC is mainly secreted and expressed by pathogenic MRSA, and has a certain specificity. At present, there is no combination of the three subunits of ClfA, IsdB and mSEC or the fusion protein of the active functional fragments of the three subunits as an immunogen. Sexual materials, the report of preparing MRSA polyvalent vaccine

Method used

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  • Methicillin-resistant staphylococcus aureus (MRSA) recombinant multivalent subunit genetic engineering vaccine and method for preparing same
  • Methicillin-resistant staphylococcus aureus (MRSA) recombinant multivalent subunit genetic engineering vaccine and method for preparing same
  • Methicillin-resistant staphylococcus aureus (MRSA) recombinant multivalent subunit genetic engineering vaccine and method for preparing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Cloning of embodiment 1 methicillin-resistant Staphylococcus aureus ClfA, IsdB, mSEC gene

[0092] 1. Methicillin-resistant Staphylococcus aureus WHO-2 (preserved by the Department of Pharmacology, Third Military Medical University)

[0093] 2. Take out the stored methicillin-resistant Staphylococcus aureus WHO-2 strain from the liquid nitrogen tank, spread it on the WHO-2 special solid medium, and cultivate it overnight at 37°C. The Genome Extraction Kit extracts the MRSA genome.

[0094] 3. The coding genes of CIfA, IsdB and mSEC were respectively amplified from the MRSA genome by PCR method.

[0095] 1) The primers were designed and synthesized as follows (the base sequence of the restriction site is underlined, and the base sequence of the linker is shown in gray)

[0096] According to the gene sequence published by GenBank and the principles of primer design, the corresponding primers were designed and restriction sites were introduced.

[0097] ClfA P1 5′TCG G...

Embodiment 2

[0175] Example 2 ClfA 484-559 Fusion gene ClfA with mSEC 484-559 --Design and synthesis of primers for mSEC(CS)

[0176] ClfA 484-559 P1 5'

[0177] -TCG GGATCC ATGACAACACCATATATTGTAGTTGTTA-3′

[0178] Bam H I

[0179] P2 5′-TGAACCGCCTCCACCCCTCTGGAATTGGTTC-3′

[0180] mSEC P3 5′-GGTGGAGGCGGTTCAATGAAGTTATTTGCT-3′

[0181] P4 5′-TTTTTTGGTTAAATGAACTTCTACATTA-3′

[0182] Using the ClfA and mSEC recovered in Example 1 as templates, respectively amplify ClfA with P1 and P2, P3 and P4 primers 484-559 and mSEC gene, the PCR amplification system is: template DNA 1μl; 10×PCR buffer 5μl; dNTPs (10mmol / L) 4μl; primers (0.025mmol / L) 1μl; Taq DNA polymerase (5u / μl) 0.5μl ; Add deionized water to a final volume of 50 μl.

[0183] The reaction system was mixed evenly, and after centrifugation, 30 μl of paraffin oil was added. Pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds...

Embodiment 3

[0186] Example 3 CS and IsdB 337-462 fusion gene CS-IsdB 337-462 (CSI) Acquisition

[0187] Primer design and synthesis

[0188] Add P1 5′-TCG to CS GGATCC ATGACAACACCATATATTGTAGTTGTTA-3′

[0189] Linker Bam H I

[0190] P5 5′-TGAACCGCCTCCACCTTTTTTGGTTAAATG-3′

[0191] IsdB 337-462 P6 5′-GGTGGAGGCGGTTCACCAACAAATGAAAAAATG-3′

[0192] P7 5′-GCG CTCGAG AGATTTATCGGTATTGGCTTTTGTA-3′

[0193] wxya

[0194] Respectively using the IsdB recovered in Example 1 and the CS recovered in Example 2 as templates, using P6 and P7, P1 and P5 as primers to amplify the IsdB with Linker respectively 337-462 and CS genes. The PCR amplification system is: template DNA 1μl; 10×PCR buffer (containing magnesium chloride) 5μl; dNTPs (10mmol / L) 4μl; primers (0.025mmol / L) 1μl; Taq DNA polymerase (5u / μl) 0.5μl , add deionized water to a final volume of 50 μl.

[0195] The reaction system was mixed evenly, and after centrifugation, 30 μl ...

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Abstract

The invention discloses methicillin-resistant staphylococcus aureus (MRSA) recombinant multivalent subunit genetic engineering vaccine and a method for preparing the same. The recombinant multivalent subunit genetic engineering vaccine is prepared by recombining, fussing and expressing the active and functional fragments of two antigen molecules of ClfA and IsdB and the specific enterotoxin C mutant for the MRSA to construct recombinant multivalent genetic engineering antigen protein under the assistance of aluminum adjuvant. The recombinant multivalent subunit genetic engineering vaccine is constructed with a unique method and prepared with simple process, is easy to be amplified and has good repeatability and high protein purity. The experiment on the animal shows that the recombinant multivalent subunit genetic engineering vaccine can effectively stimulate the body to produce higher humoral immune response and have good immune protection function.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and relates to a genetic engineering vaccine used for immunity and treatment of human methicillin-resistant Staphylococcus aureus infection and a preparation method for a multivalent subunit vaccine thereof. Background technique [0002] Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive coccus that can infect any part of the human body. Local infection persists for a long time, and the mortality rate of systemic infection is as high as 20%. It was first discovered in 1961 , has become one of the pathogenic bacteria with the highest infection rate in ICU wards, burns, and war wounds in the world. In 2005, MRSA infection surveillance data in the United States showed that there were more than 94,000 severe infections and 19,000 fatal cases in the United States throughout the year, which even surpassed the number of AIDS-related deaths. According to the survey results of China C...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/085C12N15/62A61P31/04
Inventor 周维英邹全明周红石云陈晓红郭刚毛旭虎向云潘夕春李斌李军蔡昌芝
Owner CHENGDU OLYMVAX BIOPHARM
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