Receptor selectivity lymphotoxin derivates

A lymphotoxin, selective technology for protein engineering and pharmaceutical applications

Active Publication Date: 2011-09-21
上海复旦张江生物医药股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] However, there is currently no satisfactory lymphotoxin with high selectivity for TNFRI

Method used

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  • Receptor selectivity lymphotoxin derivates
  • Receptor selectivity lymphotoxin derivates
  • Receptor selectivity lymphotoxin derivates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1 Wild-type LT, LT 24-171 , LT 28-171 preparation of

[0084] Expression vector construction:

[0085] According to the human LT sequence (BAA00064) in GENBANK, Shanghai Sangon Biotechnology Service Co., Ltd. was commissioned to synthesize the wild-type LT encoding the 1st to 171st, 24th to 171th, and 28th to 171th positions of the LT mature protein. , LT 24-171 , LT 28-171 Amino acid sequence, and cloned on the NdeI and HindIII sites of the pET32a (+) vector (purchased from Novagen), to obtain recombinant expression plasmids LT / pET32a (+), LT 24-171 / pET32a(+), LT 28-171 / pET32a(+).

[0086] Expression in E. coli:

[0087] Recombinant expression plasmid LT / pET32a(+), LT 24-171 / pET32a(+), LT 28-171 / pET32a(+) were transformed into Escherichia coli BL21(DE3), and the transformation liquid was coated on LB (containing ampicillin 100ng / μl) plate. Pick a single clone from the transformation plate and inoculate it in 1 mL of LB medium. After culturing at 3...

Embodiment 2

[0091] The preparation of embodiment 2 mutant rhLT008

[0092] Construction of rhLT008 mutant expression vector:

[0093] (1) PCR amplification mutant gene:

[0094] to LT 24-171 / pET32a(+) plasmid was used as a template, and LT-P32U and Q107E-R were used as a pair of primers to amplify to obtain the LT008-AB fragment. CD fragments. Then, LT008-AB and LT008-CD were mixed as a template, and LT-P32U and LT-P32D were used as a pair of primers to amplify to obtain the LT008 fragment containing the Q107E mutation.

[0095] LT-P32U: 5' ACACATATGATG CAC TCT ACC CTG AAA CCG 3' (SEQ ID NO: 4)

[0096] LT-P32D: 5' TGCAAGCTTCTA CAG AGC GAA GGC TCC AAA 3' (SEQ ID NO: 5)

[0097] Q107E-F: 5' CTCTTCTCCTCCGAATACCCCTTC 3' (SEQ ID NO: 6)

[0098] Q107E-R: 5'GAAGGGGTATTCGGAGGAGAAGAG 3' (SEQ ID NO: 7)

[0099] After purification, the PCR product was ligated with the pMD-18T vector, transformed into Escherichia coli DH5a, extracted the LT008 / pMD-18T plasmid, and sequenced it in forward and...

Embodiment 3

[0105] Embodiment 3 Preparation of LT006

[0106] to LT 24-171 / pET32a(+) plasmid was used as a template, and LT-P32U and S106E-Q107E-R were used as a pair of primers to amplify to obtain the LT006-AB fragment, and at the same time, LT-P32D and S106E-Q107E-F were used to amplify , to obtain the LT006-CD fragment. Then LT006-AB and LT006-CD were mixed as a template, and LT-P32U and LT-P32D were used as a pair of primers to amplify to obtain the full-length LT006 fragment containing the S106E-Q107E mutation.

[0107] The PCR primer sequences are:

[0108] S106E-Q107E-F: 5' CTCTTCTCCGAAGAATACCCCCTTC 3' (SEQ ID NO: 8)

[0109] S106E-Q107E-R: 5'GAAGGGGTATTCTTCGGAGAAGAG 3' (SEQ ID NO: 9)

[0110] Cloning, expression, and purification of LT006 were carried out in the same manner as in Example 2 to obtain 3.1 mg of LT006 protein.

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Abstract

This invention discloses some receptor-selective lymphotoxin (LT) mutants and the ratio, the ability of these lymphotoxin mutants combining with the human TNFRI to that with the human TNFRII, is more than 10. The preparation method and the use of the above said LT mutants are also disclosed. In this invention, the LT mutants bind to the TNFRI selectively and that decreases the binding to the TNFRII in a large range, thereby the toxic effect which may be caused by the TNFRII will be reduced.

Description

technical field [0001] The invention belongs to the field of protein engineering and pharmacy, and specifically relates to a receptor-selective lymphotoxin derivative and its preparation method and application. Background technique [0002] Lymphotoxin a (LTα), also known as TNFβ, is a member of the TNF superfamily and an important class of cytokines. Compared with TNF, LTα is similar in structure and function to TNF, has the same receptors TNFRI, TNFRII, LTα inhibits slightly less tumor cell types than TNF, and has different killing effects on certain tumor cells. [0003] LTα is produced by active lymphocytes and is a 25KD secreted glycoprotein. The LT precursor contains 205 amino acid residues, of which 34 amino acid residues encode a signal peptide. The mature LT has 171 amino acid residues (18kD) ( SEQID NO: 3), without a disulfide bond, the 62nd position is an N-linked glycosylation site (glycosylation is not necessary for its cytotoxic activity), and the interaction ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/525A61P33/00C12N15/28A61P35/00A61K38/19
CPCC07K14/5255A61K38/00A61P33/00A61P35/00
Inventor 刘彦君杨彤沈毅珺吴劲松曹峰王征谭靖伟吴芳许燕王海波
Owner 上海复旦张江生物医药股份有限公司
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