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PLA2R, C1q and THSD7A fusion protein and construction method and application thereof

A PLA2R, fusion protein technology, applied in DNA/RNA fragments, fusion peptides, chemical instruments and methods, etc., can solve the problems of missed detection, false detection, poor sensitivity and specificity of protein detection, and reduce missed detection and false detection. , the effect of reducing cost and time, and improving sensitivity and specificity

Active Publication Date: 2020-12-22
苏州携创生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although THSD7A protein and PLA2R protein can be used to detect serum, urine PLA2R antibody and THSD7A antibody, IMN can be diagnosed, but the detection sensitivity and specificity of a single protein are poor, resulting in missed and false detections

Method used

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  • PLA2R, C1q and THSD7A fusion protein and construction method and application thereof
  • PLA2R, C1q and THSD7A fusion protein and construction method and application thereof
  • PLA2R, C1q and THSD7A fusion protein and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Synthesis of fusion gene (target gene):

[0043] PLA2R protein (GeneID: 22925) sequence selection is as follows:

[0044]MLLSPSLLLLLLLGAPRGCAEGVAAALTPERLLEWQDKGIFVIQSESLKKCIQAGKSVLTLENCKQANKHMLWKWVSNHGLFNIGGSGCLGLNFSAPEQPLSLYECDSTLVSLRWRCNRKMITGPLQYSVQVAHDNTVVASRKYIHKWISYGSGGGDICEYLHKDLHTIKGNTHGMPCMFPFQYNHQWHHECTREGREDDLLWCATTSRYERDEKWGFCPDPTSAEVGCDTIWEKDLNSHICYQFNLLSSLSWSEAHSSCQMQGGTLLSITDETEENFIREHMSSKTVEVWMGLNQLDEHAGWQWSDGTPLNYLNWSPEVNFEPFVEDHCGTFSSFMPSAWRSRDCESTLPYICKKYLNHIDHEIVE。

[0045] THSD7A protein (GeneID: 221981) sequence selection is as follows:

[0046] AAQGEAEAPTLYLWKTGPWGRCMGDECGPGGIQTRAVWCAHVEGWTTLHTNCKQAERPNNQQNCFKVCDWHKELYDWRLGPWNQCQPVISKSLEKPLECIKGEEGIQVREIACIQKDKDIPAEDIICEYFEPKPLLEQACLIPCQ.

[0047] The C1q protein sequence (GeneID:712) is selected as follows:

[0048] GRPGRRGRPGLKG.

[0049] S1. Connect PLA2R, THSD7A, and C1q nucleotide sequences in sequence to obtain the target gene, and repeat C1q three times. Among them, a Linker sequence is ...

Embodiment 2

[0052] Construction of recombinant plasmids containing fusion genes:

[0053] 2.1 The PCR fragment obtained in Example 1 is connected with the carrier pcDNA3.1(+ / -)A (plasmid) through restriction endonuclease BamH I and Not I double digestion to obtain a recombinant plasmid;

[0054] 2.2 Transform the ligation product (recombinant plasmid) into competent Escherichia coli DH5α. The volume should not exceed 10% of the competent cells. Gently swirl the contents several times to mix the contents. Place the tube in a 42°C water bath for 30 minutes. Timed heat shock for 60 seconds, quickly transfer the tube to an ice bath for 120 seconds to cool the cells, add 400 μL LB medium to each tube, and shake slowly at 37°C for 60 minutes to revive the bacteria and express the antibiotic resistance marker gene encoded by the plasmid, at low speed Centrifuge for 2 minutes, remove the supernatant, leave about 100 μL of medium in the centrifuge tube, resuspend the bacteria, and spread the bacte...

Embodiment 3

[0057] Expression of fusion protein

[0058] 3.1 Transform the recombinant plasmid obtained in Example 2 into DH10 Bac E.coli competent cells, the volume should not exceed 5% of the competent cells, and gently rotate several times to mix the contents;

[0059] 3.2 Ice bath for 30 minutes; put the tube in a 42°C water bath, and heat shock for 90 seconds at regular intervals; quickly transfer the tube to the ice bath for 120 seconds to cool the cells; add 800 μL LB medium to each tube, and shake slowly for 4 hours at 37°C. Resuscitate the bacteria and express the antibiotic marker gene encoded by the plasmid; spread 30 μL of the bacterial solution on the resistance agar plate with a glass spreader; place the plate upside down in a constant temperature incubator at 37°C, blue and white spots will appear after 30-48 hours Colony;

[0060] 3.3 Pick a single positive white spot colony and inoculate 5mL of resistant LB, shake slowly for 12-16h, take the bacterial solution for PCR id...

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Abstract

The invention discloses a PLA2, C1q and THSD7A fusion protein and a construction method and application thereof. The construction method comprises the following steps that S1, PLA2R, THSD7 and C1q nucleotide sequences are sequentially connected to obtain a target gene, C1q is repeated three times, Linker sequences are added between PLA2R and THSD7A, Linker sequences are added between THSD7 and C1q, and Linker sequences are added between adjacent C1q; S2, an eukaryotic KOZAK sequence is added before the target gene, a restriction endonuclease site Bam I is added at the upstream, a Not I enzymedigestion site is added at the downstream, a 6* His tag sequence is added at the C end, and a complete sequence gene is designed according to mammalian cell preference; S3, the complete sequence geneobtained in the step S2 and plasmids are subjected to double enzyme digestion to obtain an enzyme digestion product; S4, the enzyme digestion product obtained in the step S3 is connected through T4 ligase to obtain recombinant plasmids; S5, the recombinant plasmids are expressed in mammalian cells to obtain the PLA2R, C1q and THSD7A fusion protein. The fusion protein constructed by the method canimprove the sensitivity and specificity of a detection kit and reduce missed detection and false detection.

Description

technical field [0001] The invention relates to the technical field of biomedical engineering, in particular to PLA2R, C1q, THSD7A fusion protein and its construction method and application. Background technique [0002] Membranous Nephropathy (MN) is the most common pathological type of nephrotic syndrome in adults. Today, the pathological diagnosis technology of various medical centers continues to mature, and the incidence and diagnosis rate of MN are also increasing year by year, and it has become the most common type of nephrotic syndrome. cause of disease. Its pathological feature is that the pathogenic target antigen binds to the antibody and deposits in the glomerular podocytes, resulting in thickening of the glomerular basement membrane, loss of podocyte foot processes, and reactivation of complement to cause damage. MN is divided into 3 types according to the etiology and pathological features, including Idiopathic Membranous Nephropathy (IMN), Secondary Membranou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10G01N33/68
CPCC07K14/4713C07K14/7056C07K2319/00C12N15/85C12N2800/22G01N33/6893G01N2800/347
Inventor 秦枫蒋洪娇周志琼颜松张小飞周玥黄敬双张伟万文琴鲜静吴斌
Owner 苏州携创生物技术有限公司
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