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Antineoplastic invasion transfer function of snake venom metalloprotease inhibitors BJ46a and uses thereof

A metalloprotease and inhibitor technology, applied in the field of medical biology, to achieve obvious effects of anti-tumor invasion and metastasis, prevention and treatment of invasion and metastasis

Inactive Publication Date: 2009-05-13
FUJIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Retrieval of relevant materials including Chinese patents shows that snake venom metalloproteinase inhibitor BJ46a has anti-tumor invasion and metastasis effects and has not been reported in the literature for its application in the prevention and treatment of tumors

Method used

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  • Antineoplastic invasion transfer function of snake venom metalloprotease inhibitors BJ46a and uses thereof
  • Antineoplastic invasion transfer function of snake venom metalloprotease inhibitors BJ46a and uses thereof
  • Antineoplastic invasion transfer function of snake venom metalloprotease inhibitors BJ46a and uses thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] BJ46a gene synthesis

[0041] According to the BJ46a gene sequence (AF294836) found in GenBank, it was divided into 20 fragments, and 10 pairs of primers were designed accordingly, synthesized and purified.

[0042] The 20 fragment sequences are:

[0043] 1: 5’-ATG AAT TCC CTG GTAGCT CTC GTG CTC CTG GGT CAG ATTATA GGA TCTACG CTT AGC TCT CAAGTG AGG G-3’

[0044] 2: 5’-ACC TGT ATC TGT CCA GTC TTT AGC GTC TTT CTC GTC GCATTC TAA ATCCCC CCT CAC TTG AGA GCT AAG C-3’

[0045] 3: 5’-GAG TGG ACA GAT ACA GGT GTG CGC TAC ATC AAC GAG CAT AAA CTA CATGG ATAC AAA TAT GCC CTCAAT GTA A-3’

[0046] 4: 5’-TTA AGG AAG ACT GCC ACC CAA TCG CCA TCC CAG GGA ACG ACA ACG ATATTC TTA ATT ACA TTG AGG GCA TAT TTG-3’

[0047] 5: 5’-TGG GTG GCA GTC TTC CTT AAA TTA AAT CTT CTG GAG ACA GAA TGT CATGTG TTG GAT CCA ACT CCT GTC A-3’

[0048] 6: 5’-ACA GTC CAT TTC AAC AGC ATG ATT ATG CTG TGG CCT TAC AGT ACA ATTCTT GAC AGG AGT TGG ATC CAA C-3’

[0049] 7: 5’-ATG CTG TTG AAA TGG ACT GTG ATG TCA AGA TAA TG...

Embodiment 2

[0086] Correction of wrong bases by GeneTailor site-specific mutagenesis system

[0087] Sequence determination after synthesis of BJ46a showed that it was basically consistent with the gene sequence of snake BJ46a, but there were errors in 6 sites. According to the sequencing results, the wrong positions were corrected one by one by the method of site-directed mutagenesis. Each site mutation consists of three steps: first, use a site-specific methylase to methylate the target DNA; then conduct a mutation reaction through two overlapping primers, one of which carries the target mutation site; finally The mutant product was transformed into mcrBC+ Escherichia coli DH5α-T1, the clone was picked, identified by SacI and XhoI double enzyme digestion, and the desired mutation was confirmed by sequencing. Extract the plasmid containing the mutated gene, repeat three steps to complete another site mutation, and finally obtain the correct target gene.

Embodiment 3

[0089] Construction of BJ46a Baculovirus Expression Vector

[0090] BJ46a was inserted into the MCS of the baculovirus transposable vector pFastBac HTC after double digestion at the SacI and XhoI sites, transformed into Escherichia coli DH5α-T1, positive recombinants were screened with LB / AP plate, PCR amplification, enzyme digestion identification , extract pFastBac HT C—BJ46a recombinant to further transform DH10Bac competent cells, after 48 hours, pass blue-white screening, pick a single white colony and streak, confirm that the obtained colonies are all white, and pick clones for PCR identification. The results showed that the baculovirus recombinant transposable vector and the recombinant shuttle vector were successfully constructed. The high-purity plasmid Bacmid-BJ46a recombinant used for cell transfection was extracted.

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Abstract

The invention discloses a snake venom metal protease inhibitor BJ46a capable of resisting attack and transference of tumor and application thereof, and belongs to the field of medical organism. In the invention, according to the BJ46a gene sequence (AF294836) in GenBank, the BJ46a full gene is designed and synthesized. An expression vector cloned to baculovirus can generate the recombined BJ46a protein capable of inhibiting the activity of the substrate metal protease in Sf9 insect cells, and in vivo and in vitro experiments show that the BJ46a protein has the functions of resisting the attack and transference of melanoma cells B16. The inhibitor adopts the genetic transfection technique, and can establish B16 / pcDNA3.1HisC-BJ46a cell strains with stable transfection, and the in vivo and in vitro experiments prove that the BJ46a can inhibit the attack and transference of B16 cells at the gene level. The inhibitor is applied to preventing and treating the attack and transference of tumor, and has great application prospect.

Description

technical field [0001] The invention relates to the field of medical biology, in particular to a snake venom metalloproteinase inhibitor BJ46a anti-tumor invasion and metastasis effect and application. Background technique [0002] The incidence of malignant tumors has been increasing year by year worldwide since the 1970s, and ranks second among the causes of death in my country. In my country, 1.8 million people are newly diagnosed with cancer and 1.5 million people die of cancer every year. Malignant tumors not only bring mental and physical pain to the patients themselves and their relatives, but also put enormous pressure on my country's limited health resources. The control and treatment of malignant tumors is one of the major public health problems faced by our government. [0003] Uncontrolled proliferation of tumor tissue, abnormal differentiation of tumor cells, and the ability of tumor cells to invade and metastasize are the most basic biological characteristics...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/15C12N15/79C07K14/81A61K38/57A61K48/00A61P35/04
Inventor 林建银林旭徐剑文
Owner FUJIAN MEDICAL UNIV
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