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Fusion expression protein as well as preparation method and application thereof

A technology of fusion protein and expression vector, which is applied in the field of fusion expression protein and its preparation, can solve the problems of high cost, complex preparation method of composite quality control products, inconvenient operation, etc., achieve low cost, improve detection convenience, and good stability and accuracy effects

Pending Publication Date: 2022-04-15
巴迪泰(广西)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

[0010] The technical problem to be solved by the present invention is: the quality control products for inflammation items used in the existing inflammation diagnosis and detection are either single-item quality control products, which are inconvenient to operate, or the preparation method of composite quality control products is complicated and the cost is high.

Method used

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  • Fusion expression protein as well as preparation method and application thereof
  • Fusion expression protein as well as preparation method and application thereof
  • Fusion expression protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 synthetic target gene

[0051] Query the complete CDS region of 4 genes in NCBI, among which, the nucleotide sequences of CRP gene, SAA gene, PCT gene and IL6 sequence are respectively as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:3, SEQ ID NO: ID NO:4 shown.

[0052] Insert Hind III restriction endonuclease cutting site and Kozak sequence before CRP initiation codon ATG, wherein the nucleotide sequence of Hind III restriction endonuclease cutting site is as shown in SEQ ID NO:5 ( AAGCTT), the nucleotide sequence of Kozak is shown in SEQ ID NO: 6 (GCCACCATGG), the purpose of inserting the Kozak sequence among the present invention is to ensure that the target gene transcript can be effectively translated.

[0053] Insert the 6*His tag before the IL6 stop codon TAG, the nucleotide sequence is as shown in SEQ ID NO:7 (CATCATCATCATCATCAC), and insert the Xba I restriction site after the TAG, the nucleotide sequence is as SEQ ID NO:8 Shown (TCTAGA). At...

Embodiment 2

[0056] The construction of embodiment 2 recombinant vector

[0057] The target gene synthesized in Example 1 and the mammalian expression vector pcDNA3.1 were simultaneously digested with Hind III and Xba I restriction endonucleases for 30 min, and the enzyme digestion system is shown in Table 1 below. The digested products were detected by agarose gel electrophoresis and recovered for later use.

[0058] Table 1 enzyme digestion system

[0059] Reagent Volume / μL pcDNA3.1 / recombination target gene 3.6 / 2.6 10×Cut buffer 4 Hind III 1.5 Xba I 1.5 sterile water Make up to 40μL

[0060] The recovered target gene fragment was ligated with pcDNA3.1 at 6 times the mass ratio of the vector with T4 DNA ligase at 16°C overnight, and the ligated product was transformed into Top 10 competent cells, and plated on LB containing 50 μg / mL ampicillin Solid culture medium, cultured upside down in a constant temperature incubator at 37°C for 12-16 ...

Embodiment 3

[0062] Expression and identification of embodiment 3 fusion protein

[0063] (1) After further culturing the recombinant vector successfully obtained in Example 2, use an endotoxin-free plasmid extraction kit to obtain a large number of recombinant plasmids for future use.

[0064] (2) Resuscitate HEK 293T cells, culture the cells after subculture until the cells grow to a density of about 70-80%, and then transfect.

[0065] (3) Dilute the plasmid and Lipofectamine 2000 transfection reagent in Opti-MEM and let it stand for 5 minutes. After mixing the plasmid and transfection reagent, let it stand for 30 minutes, add it evenly to the cell culture dish, and culture in the cell incubator.

[0066] (4) After the cells have been cultured for 72 hours, take out the cells from the incubator, wash the residual medium with PBS, lyse the cells with RIPA lysate (containing protease inhibitors and PMSF), scrape the cells with the thick part of a yellow pipette, and then suck out the liqu...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a fusion expression protein as well as a preparation method and application thereof. An inflammation item quality control product adopted during existing inflammation diagnosis and detection is either a single-item quality control product and is inconvenient to operate, or a composite quality control product preparation method is complex and high in cost, aiming at the problems, the invention provides a fusion expression protein, four inflammation marker proteins of RP, SAA, PCT and IL6 are creatively subjected to fusion expression, and the fusion expression protein is used for preparing the inflammation marker protein. The obtained fusion protein can be used for respectively preparing PCT and IL6 project composite quality control and CRP and SAA project composite quality control according to the proportion, not only has good stability and accuracy, but also can effectively reduce the cost of quality control products, improve the convenience of in-vitro diagnosis and detection and well control the batch-to-batch difference of the quality control products.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a fusion expression protein and its preparation method and application. Background technique [0002] Infectious diseases are common clinical diseases and frequently-occurring diseases. They are mainly caused by inflammatory reactions caused by bacteria, viruses and fungi invading the body, and most of them are accompanied by fever. Infectious diseases caused by pathogens such as bacteria, viruses and fungi and their products are still one of the main causes of human death and disability, threatening human health. Therefore, the diagnosis and detection of inflammation is particularly important. [0003] In the diagnosis and detection of inflammation, it is particularly important to select appropriate markers of inflammatory response. [0004] Since Assicot et al first reported that the level of procalcitonin (PCT) was significantly increased in patients with s...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N15/62C12N5/10G01N33/68G01N33/74
CPCC12N15/85C07K14/4737C07K14/775C07K14/585C07K14/5412C12N5/0686G01N33/6893G01N33/74C12N2800/107C12N2840/105C07K2319/21C07K2319/00C12N2510/02G01N2800/7095G01N2333/4737G01N2333/775G01N2333/585G01N2333/5412
Inventor 王洪涛梁亚运王怀忠李康梁刚李登红
Owner 巴迪泰(广西)生物科技有限公司
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