Recombinant staphylococcus aureus ClfA protein vaccine and construction method of eukaryotic expression engineering cell line of protein vaccine

A staphylococcus and protein vaccine technology, applied in other methods of inserting foreign genetic materials, recombinant DNA technology, genetic engineering, etc., can solve the problems of difficult expression and purification of protein products, and achieve single antigenic characteristics, strong feasibility, The effect of design science

Inactive Publication Date: 2017-04-26
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Another object of the present invention is to provide a method for constructing eukaryotic expression engineering cell lines to solve the problem that some protein products are difficult to express and purify effectively in the existing vaccine research and development process

Method used

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  • Recombinant staphylococcus aureus ClfA protein vaccine and construction method of eukaryotic expression engineering cell line of protein vaccine
  • Recombinant staphylococcus aureus ClfA protein vaccine and construction method of eukaryotic expression engineering cell line of protein vaccine
  • Recombinant staphylococcus aureus ClfA protein vaccine and construction method of eukaryotic expression engineering cell line of protein vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1. The construction method of eukaryotic expression engineering cell line is as follows:

[0034] Step 1. According to the genome sequence of Staphylococcus aureus published online by NCBI, design ClfA gene primers; wherein, the upstream primer ClfA-1168F1, the sequence is GACA AAGCTT AATATGGGCGAAACGAGTG, the downstream primer ClfA-1168R1, the sequence is CTCGAG ATCAATTTGGATTTGGGAA.

[0035] Step 2, taking the genomic DNA of Staphylococcus aureus (coded as MD2) as a template, extracting the genomic DNA, and amplifying the ClfA gene sequence with primers ClfA-1168F1 and ClfA-1168R1.

[0036] The extraction method for the genomic DNA of Staphylococcus aureus is:

[0037] 1. Cultivate 3ml of bacterial culture overnight, collect the bacterial cells by centrifugation at 13000g at room temperature for 1min, resuspend the bacterial cells with 160μl Buffer EB, then add 40μl lysozyme (100mg / ml), vortex and mix, place at 37°C for 30min; centrifuge at 5000g 3min and...

Embodiment 2

[0155] The difference between this example and Example 1 is that the double enzyme digestion system of the ClfA gene and the carrier pcDNA 3.1 V5-His B in step 3 is 50 μl, and the enzyme digestion reaction condition is overnight enzyme digestion at 2-8°C.

[0156] The double enzyme digestion reaction system involved is:

[0157]

[0158] Pipette well, then add restriction endonuclease

[0159] Hind III (10u / μl) 0.5μl

[0160] Xho I (10u / μl) 0.7μl

[0161] Pipette evenly with a 10μl pipette, cover the cap of the centrifuge tube, and centrifuge briefly to fully mix the components; digest overnight at 2-8°C.

[0162] The ligation reaction system of ClfA and pcDNA 3.1 V5-His B after enzyme digestion is 20 μl, and the reaction conditions are 16°C overnight ligation reaction system and reaction conditions (1.5mL centrifuge tube):

[0163]

[0164] Connect overnight at 16°C for 8-16 hours, generally you can get better results.

[0165] In step 6, when using Ni-NTA affinity ...

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Abstract

The invention discloses a recombinant staphylococcus aureus ClfA protein vaccine and a construction method of a eukaryotic expression engineering cell line of the protein vaccine, and belongs to the field of biological pharmacy. The gene sequence of staphylococcus aureus ClfA is SEQ ID NO. 1. The construction method mainly includes the steps: separating and identifying the staphylococcus aureus; extracting genome DNA (deoxyribonucleic acid) of the staphylococcus aureus; designing a primer amplification ClfA gene sequence by taking the extracted staphylococcus aureus genome DNA as a template; connecting the ClfA sequence with pEASYR-T1 Simple Cloning Vector and performing amplification on DH5 alpha; extracting plasmids and respectively performing double enzyme digestion and connection for the ClfA and pcDNA 3.1 V5-His B to obtain recombinant plasmids ClfA-pcDNA 3.1 V5-His B; converting connection recovery products into Escherichia coli DH5 alpha; screening positive clones and extracting the recombinant plasmids ClfA-pcDNA 3.1; transfecting human breast cancer cells MCF-7 by the aid of lipidosome 2000 (lippo2000); screening positive cell clones and high expressing clones by G-418. The construction method can solve the problem that protein products easily form inclusion bodies and are not easily purified when procaryotic organism expression long-sequence genes encode high-molecular-weight protein products.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a recombinant Staphylococcus aureus ClfA protein vaccine and a method for constructing eukaryotic expression engineering cell lines thereof. Background technique [0002] Staphylococcus aureus causes a variety of infections in humans and animals worldwide. In humans, it mainly causes local suppurative infection, pneumonia, enteritis, pericarditis and other local or systemic infections. It is also one of the main pathogenic bacteria that cause food poisoning in humans; In veterinary clinics, Staphylococcus aureus often causes systemic or local infections such as mastitis, exudative dermatitis, urinary tract infection, meningitis, and endocarditis in various animals. Because clinically isolated Staphylococcus aureus strains are often resistant to a variety of antibiotics, not only increases the cost of clinical medication, but also the emergence of strains resistant to...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/88C12N5/10A61K39/085A61P31/04
CPCC07K14/31A61K39/085C12N15/88
Inventor 赵兴绪武小虎张全伟马友记张勇
Owner GANSU AGRI UNIV
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