Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof

A technology of recombinant protein and staphylococcus, applied in the biological field, can solve the problems of few treatment methods and difficult control of the situation, and achieve the effect of easy separation and purification, simple steps, and good immune protection

Active Publication Date: 2013-03-20
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And as the multi-antibiotic resistance of the bacteria makes the situatio

Method used

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  • Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof
  • Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof
  • Recombinant protein HF2 used for methicillin-resistant staphylococcus aureus (MRSA) vaccine, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1: The DNA sequence of methicillin-resistant Staphylococcus aureus Hla (H35L delta110-150) was synthesized by Shanghai Jierui Biotechnology Co., Ltd.

Embodiment 2

[0055] Example 2: PCR method was used to amplify the H portion used to construct HF2.

[0056] 1. Considering the connecting peptide SEQ ID NO:3 of the H segment, design the following primers to clone the H gene from the synthetic DNA sequence:

[0057] SEQ ID NO: 7gc ggatcc at ggcagattct gatattaataataa (underlined restriction restriction site BamHI)

[0058] SEQ ID NO:8aactaagctgccaccgccac catttgtcat ttc

[0059] 2. PCR clone gene

[0060] PCR system:

[0061] Template (100ng / μl) 0.5μl SEQ ID NO: 7 (50 μM) 1μl SEQ ID NO:8 (50μM) 1μl Primerstar Taq Enzyme 0.5μl dNTP 4μl 5X Buffer 10μl Sterilized double distilled water 33μl total capacity 50μl

[0062] The PCR amplification reaction conditions were denaturation at 98°C for 10s, annealing at 60°C for 10s, extension at 72°C for 1min and 30s, 30 cycles, and complete extension at 72°C for 6min. After the reaction was completed, the PCR amplification results wer...

Embodiment 3

[0064] Example 3: Cloning of Methicillin-resistant Staphylococcus aureus Fibronectin Binding Protein A Active Fragment FNBA2 Active Fragment (F2)

[0065] 1. First, according to the full-length gene sequence of MRSA-252FnBA protein, use bioinformatics software for structural analysis to determine the FnBA1 target gene fragment that needs to be amplified.

[0066] 2. According to the analysis results, the PCR method was used to amplify the FnBA1 target gene fragment from the MRSA-252 genome, and the amplification steps were as follows:

[0067] 1) Design PCR primers as follows, respectively SEQ ID NO: 9-10

[0068] SEQ ID NO: 9ggtggcggtg gcagcttagt ttatatagt aataaagc

[0069] SEQ ID NO:10ttttcctttt gcggccgc ct aacctttgtt tgttgattct tctc (the restriction site NotI is underlined)

[0070] 2) The methicillin-resistant Staphylococcus aureus MRSA-252 strain was taken out of the -80°C freezer and spread on the MRSA-252 special solid medium, cultivated overnight at 37°C, and then ...

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Abstract

The invention discloses a recombinant fusion protein HF2 which is composed of staphylococcus aureus alpha hemolysin (H1a) and a fibronectin binding protein (FNBA) segment, wherein the H1a and the FNBA are fused through a linker peptide. The protein can be used for preparing subunit vaccines and relevant detection kits capable of resisting infections of the methicillin-resistant staphylococcus aureus (MRSA). A gene engineering technology is used to recombine and express the fused protein. The method has high expression level, facilitates separation and purification and has high efficiency and safety. Animal experiment results show that the recombinant fusion protein HF2 can effectively stimulate a body to produce relatively high humoral immune response and good immune protection effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to a recombinant protein HF2 of a methicillin-resistant Staphylococcus aureus alpha hemolysin nontoxic mutant and an active fragment of fibronectin-binding protein A, and the invention further relates to a preparation method of the recombinant protein and its application in preparing vaccines and detection kits. Background technique [0002] Staphylococcus aureus can cause a wide range of diseases in the human body, such as bacteremia, endocarditis, folliculitis, boils, boils, carbuncles, impetigo, cellulitis, staphylomycosis, toxic shock syndrome Syndrome, central system infection, infectious eye inflammatory disease, osteomyelitis, bone and joint infection, respiratory system infection, etc., pose a great threat to human health. And because the multi-antibiotic resistance of the bacteria makes the situation more difficult to control, the options for treatment are fewer and...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21A61K39/085A61P31/04G01N33/569C12R1/19
Inventor 冯强邹全明曾浩樊绍文卢陆董衍东鲁东水吴翼蔡昌芝
Owner CHENGDU OLYMVAX BIOPHARM
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