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tsm1 polypeptide and its application in the preparation of anti-complement drugs

A 1.TSM1, peptide technology, applied in the fields of genetic engineering and biomedicine, can solve the problems of difficult oral administration, poor specificity, and decreased anti-infection ability

Active Publication Date: 2020-07-24
HUBEI UNIVERSITY OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that although immunosuppressant drugs widely used in clinical practice, such as glucocorticoids, cyclosporine, and macrolide antibiotics, have certain therapeutic effects on some diseases related to excessive activation of complement, they are not Specific complement inhibitors have poor specificity and are difficult to take orally. Long-term use will reduce the body’s immunosuppressive ability, resulting in a decline in anti-infection ability, easy secondary infection and potential spread of lesions, and various complications and side effects

Method used

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  • tsm1 polypeptide and its application in the preparation of anti-complement drugs

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Embodiment 1

[0029] Embodiment 1: Structural protein sequence analysis

[0030] 1. Use bioinformatics methods to search the three major nucleic acid databases through the Internet to obtain the tapeworm polypeptide sequence, and number the obtained sequence.

[0031] 2. Use the website http: / / www.cbs.dtu.dk / services / SignalP to analyze the sequence and remove the signal peptide sequence.

[0032] 3. According to the kunitz pattern amino acid sequence, the six cysteines are arranged regularly.

[0033] XXXXXXXX C XXXXXXXX C XXXXXXXXXXXXXXXX C XXXXXXXX C XXXXXXXXXXXX C XXX C XXXXX

[0034] Sequences with this structure can be screened out from the sequences, analyzed and compared, and finally the sequences of TSM series polypeptides and sequences that may have anti-complement functions are obtained.

[0035] 4. Amino acid sequence of TSM1 polypeptide:

[0036] KMSYINRCNLPISSGQCRGYFLRYGYDSEADECRRFVYSGCRGNRNNNFFTYNECMNRCYMRFK (SEQ ID NO. 1)

[0037] 5. The codon-optimized nucleic acid...

Embodiment 2

[0040] Embodiment 2: TSM polypeptide clone

[0041] 1. TSM1 primer design:

[0042] TSM1-1(FP3):

[0043] CTGCATATGAAAATGAGCTATATTAACCGCTGCAACCTGCCGATTAGC (SEQ ID NO. 3)

[0044] TSM1-2(FP2):

[0045] AACCTGCCGATTAGCAGCGGCCAGTGCCGCGGCTATTTTCTGCGCTAT (SEQ ID NO. 4)

[0046] TSM1-3(FP1):

[0047] TATTTTCTGCGCTATGGCTATGATAGCGAAGCGGATGAATGCCGCCGC (SEQ ID NO. 5)

[0048] TSM1-4(RP1):

[0049] GTTGCGGTTGCCGCGGCAGCCGCTATACACAAAGCGGCGGCATTCATC (SEQ ID NO. 6)

[0050] TSM1-5(RP2):

[0051] GCGGTTCATGCATTCGTTATAGGTAAAAAAGTTGTTGCGGTTGCCGCG (SEQ ID NO. 7)

[0052] TSM1-6(RP3):

[0053] GTGCTCGAGTCATTTAAAGCGCATATAGCAGCGGTTCATGCATTC (SEQ ID NO. 8)

[0054] 2. Primer preparation: Centrifuge the primer powder at 12000r for 2min, add ultrapure water to 100uM according to the instructions, centrifuge for 30s, take 10μl+90μlddH2O, centrifuge for 30s, 12000r, and store at -20°C.

[0055] 3. Synthesis of the target gene: Amplify the complete target gene sequence through 3 rounds of PCR ...

Embodiment 3

[0099] Embodiment 3: Expression and purification of TSM recombinant protein

[0100] 1. The target fragment is transformed into expression bacteria:

[0101](1) Take 2 μl of the extracted plasmid and add it to E.coli / Transetta (DE3) competent cells, mix gently, place in ice bath for 30 minutes, heat shock in 42°C water bath for 90 seconds, immediately place on ice to cool for 5 minutes, add 500 μl LB For liquid medium, put it on a constant temperature shaker at 37°C, 180r, incubate for 45min, then centrifuge at 3700r for 5min, remove the supernatant, leave an appropriate amount of supernatant, blow off the cell pellet, and spread it on an LB medium plate containing 100ug / ml Kana, at a constant temperature of 37°C Cultivate for 13h;

[0102] (2) Pick a single colony on the plate, add 500 μl of anti-Kana LB medium, shake at a constant temperature of 37 ° C, 180 r, and culture for 10.5 h to obtain expression bacteria, add 500 μl of 30% glycerol to mix and store at -20 ° C.

[0...

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Abstract

The invention discloses TSM1 polypeptide derived from pork tapeworms. According to the genetic engineering technology, primers are designed, cloned, expressed and purified to obtain TSM1 protein, andthe anticomplementary function of TSM1 is determined by a classical approach, complement hemolytic tests. The TSM1 polypeptide comprises 63 amino acids. The full length of a gene for coding the protein is 189bp. The theoretical molecular weight of the protein is 9820Da. The protein with a stable physicochemical property has three pairs of disulfide bonds, and comprises a Kunitz structure domain. The TSM1 polypeptide and the application thereof have the advantages that a foundation is laid for search for complement resistance targets and natural anticomplement drugs, new guide molecules are provided for research and development of the natural anticomplement drugs, and the TSM1 is expected to become a new natural anticomplement drug in the classical path.

Description

technical field [0001] The invention belongs to the fields of genetic engineering and biomedicine, and specifically relates to TSM1 polypeptide derived from Taenia solium and its application in preparing anti-complement drugs. Background technique [0002] Taenia solium, also known as Taenia solium, Taenia solium, Taenia solium, Taenia solium, is widely distributed, but its infection rate is not high. Taenia solium is a zoonotic parasitic worm, and the human body is the only ultimate host of the worm. Taenia solium secretes some proteases and protease inhibitors, so that it forms an immune mechanism that interacts with the human body during the long-term parasitic process of the human body, and performs immune evasion through immune isolation, molecular simulation, destructive antibodies, immunosuppression, and immune regulation. . [0003] In recent years, 199 proteases and 35 protease inhibitors have been identified from Taenia solium by using the genome database and pro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/81C12N15/15C12N15/11A61K38/57A61P37/02A61P29/00
CPCA61K38/00A61P29/00A61P37/02C07K14/8114
Inventor 陈宗运丁莉罗旭东刘田利胡芳芳
Owner HUBEI UNIVERSITY OF MEDICINE
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