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CBLB502-Fc fusion protein and preparation method thereof

A technology of CBLB502 and fusion protein, which is applied in the fields of peptide/protein components, chemical instruments and methods, hybrid peptides, etc., can solve the problems of complex purification process, difficult removal of heat source or endotoxin, and achieve simplified protein purification process and improved stability Sexuality, the effect of great application prospects

Active Publication Date: 2016-07-27
北京纽莱福生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, CBLB502 in literature reports is expressed in prokaryotes (YoonSI, KurnasovO, NatarajanV, et al. [J]. Science, 2012, 335(6070): 859-64.), and its protein is expressed in the form of inclusion bodies, resulting in purification process Complex and difficult to remove pyrogens or endotoxins

Method used

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  • CBLB502-Fc fusion protein and preparation method thereof
  • CBLB502-Fc fusion protein and preparation method thereof
  • CBLB502-Fc fusion protein and preparation method thereof

Examples

Experimental program
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Embodiment 1

[0053] The amplification of embodiment 1CBLB502 gene

[0054] Primers 502F and 502R were designed according to the CBLB502 gene sequence, and the target gene fragment was amplified by DNA polymerase PCR using the vector pUC57-CBLB502 as a template. Among them, the base sequence (5'-3') of 502F is GATATCCCTAGGCCACCATGGAA, and the restriction sites are AvrII and EcoRV; the base sequence (5'-3') of 502R is GGATCCCAGCAGGCTCAGGACG, and the restriction site is BamHI.

[0055] PCR reaction system (50 μL) includes: 40ng template DNA sample, 1.5 μL forward primer set 502F (0.3 μM), 1.5 μL reverse primer set 502R (0.3 μM), 25 μL 2×PCR buffer, 10 μL dNTP (2 mM), 1 μL KODFX DNA polymer Enzyme (1U), ddH 2 O supplemented to 50 μL.

[0056] The PCR reaction conditions included: 94°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 65°C annealing for 30 s, 68°C extension for 1 min, 30 cycles, and then 68°C extension for 7 min. The PCR amplification product was subjected to 1.0% agar...

Embodiment 2

[0057] Example 2 Construction and identification of pUC57-CBLB502-Fc plasmid

[0058] The target gene fragment and pUC57-Fc were double-digested with restriction endonucleases AvrII and BamHI, and the digested products were detected by 1.0% agarose gel electrophoresis, and the target fragment was recovered with a DNA gel recovery kit after gel cutting. Transform competent Escherichia coli after ligation with T4 DNA ligase, select positive single clones, verify and sequence with primers P57YF and P57YR. The base sequence (5'-3') of P57YF is CGCCAGGGTTTTTCCCAGTCAC, and the base sequence (5'-3') of P57YR is GCAGGACAGGGAGGACAAGTG.

[0059] The positive colony identification method is as follows: select positive monoclonal to ampicillin-resistant LB medium, shake the bacteria on a constant temperature shaker at 37°C for 6 hours, and take the bacterial liquid for PCR verification. PCR reaction system (20 μL): 1 μL bacterial liquid, 1 μL P57YF, 1 μL P57YR, 10 μL 2×TaqMasterMix, 7 μL...

Embodiment 3

[0062] Example 3 Construction and identification of pcDNA3.1-CBLB502-Fc plasmid

[0063] pcDNA3.1 and pUC57-CBLB502-Fc were digested with restriction endonucleases EcoRV and HindIII, and the CBLB502-Fc gene fragment obtained after digestion was cloned into a vector, transformed into Escherichia coli competent cells, and positive single clones were selected and used Restriction digestion was verified and sent for sequencing, and the constructed plasmid was named pcDNA3.1-CBLB502-Fc. CBLB502-Fc-specific bands can be seen at about 1800bp, such as figure 2 shown, consistent with the expected results. The obtained recombinant plasmid was sequenced, and the sequencing result was correct, and the pcDNA3.1-CBLB502-Fc plasmid was constructed successfully.

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Abstract

The invention discloses a CBLB502-Fc fusion protein and a preparation method thereof. The fusion protein comprises CBLB502 protein and an immune globulin Fc segment, wherein the Fc segment is selected from immune globulin of human or animals or subtypes thereof. The fusion protein with high activity, good stability and long half-life period is obtained by building a eukaryotic expression vector of the CBLB502-Fc fusion protein and transfecting to eukaryotic cells, and can be applied to radiation protection, remission and restoration of bone marrow and gastrointestinal tract injuries caused by chemical medicines, tumor prevention and the like.

Description

technical field [0001] The invention relates to a fusion protein, in particular to a CBLB502-Fc fusion protein and a preparation method thereof, and belongs to the field of biotechnology. Background technique [0002] Toll-like receptors (Toll-like receptors, TLRs) play an important role in the body's innate immunity and acquired immunity (AkiraS, TakedaK, KaishoT.[J].Natureimmunology,2001,2(8):675-80.), currently Thirteen TLRs have been discovered in mammalian cells. TLRs can specifically recognize pathogen-associated pattern molecules such as bacteria and viruses, and activate myeloid differentiation factor 88 (MyD88)-dependent signaling pathways to activate innate immunity (CPaulC, FaqiAS.[J].Reproductivetoxicology,2014,46(7):12 -9.), further causing inflammatory response, antiviral response and differentiation and maturation of immune cells, thereby protecting the body from the invasion of pathogens. [0003] Toll-like receptor 5 (TLR5) is a member of the TLR family, a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62A61K38/16A61K47/48A61P35/00A61P1/00A61P25/00
Inventor 胡显文段海峰陈柯言陈强张伟高丽华王友亮邵勇焦长乐谭招丽靳阳殷慧慧
Owner 北京纽莱福生物科技有限公司
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