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Dual target effected chimera recombinant, its construction method and application

A technology of effector genes and construction methods, applied in gene therapy, recombinant DNA technology, chemical instruments and methods, etc., can solve the problems of high mortality, easy metastasis, and poor prognosis

Inactive Publication Date: 2008-03-26
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical studies have shown that about 20-30% of invasive breast cancer patients have breast cancer cells with significantly amplified HER-2 / neu, highly expressed HER-2, and the expression level of metastases is consistent with that of primary cancer cells; Van Gogh Infiltrating breast cancer cases with horizontal HER-2 expression are not sensitive to chemotherapy, have poor prognosis, are prone to metastasis, and have high mortality

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Construction of SDF-1 / 54R-IgG1HCHR-Decorin chimeric gene and its prokaryotic expression system

[0071] 1. Using primer 15'-ggggtaccgacgacgacgacaagaagcccgtcagcctgagctacagat-3' and primer 25'-ccgctcgagcttcgggtcaatgcacac-3' to SDF-54 / R / pET30-a (+) (invention name "stromal cell-derived factor-1 recombinant mutant SDF-1 / Construction, preparation and application of 54R", patent application number "03146833.0") as a template to amplify SDF-54 / R, introduce KpnI and XhoI restriction sites at the 5' and 3' ends, respectively, and introduce EK at the 5' end Recognition Site-(Asp) 4 Lys coding sequence in order to excise the His-Tag of the fusion protein that is finally expressed, and remove the stop codon at the 3' end of the coding sequence at the same time. The PCR cycle was: 94°C pre-denaturation for 5 minutes, then 94°C for 10s, 55°C for 10s, 72°C for 10s, 32 cycles, and finally 72°C for 3min. The PCR product was separated by agarose gel electrophoresis to obtain a band of...

Embodiment 2

[0078] Prokaryotic Expression and Purification of SDF-54 / R-IgG1HCHR-Decorin / pET-30a(+) Chimeric Recombinant

[0079] 1. Add about 200ng of the confirmed SDF-54 / R-IgG1HCHR-Decorin / pET-30a(+) recombinant plasmid into 200ul competent E. coli BL21(DE3)(NOVAGEN) cells (prepared by calcium chloride method), gently Mix well, place on ice for 30min, then heat shock at 42°C for 90s, immediately transfer to ice and place for 2min, then add 800ul LB culture medium, shake and culture at 37°C for 45min, take 200ul and spread it on the 50ug / ml kana On LB plates of mycin, culture overnight at 37°C (about 12hr), randomly pick 5 positive colonies and inoculate them into 50ml LB liquid medium (containing 50ug / ml kanamycin) and shake at 37°C for about 12hr, then respectively Take 50ul of the above culture solution and transfer it to 50ml of LB liquid medium (containing 50ug / ml kanamycin) and shake it for about 3 hours at 37°C. When OD600=0.5, add IPTG with a final concentration of 0.75mM at 37°C...

Embodiment 3

[0096] Bioactivity assay

[0097] 1. MTT

[0098] Collect the logarithmic phase MDA-MB-231 cells, adjust the concentration of the cell suspension, inoculate in seven 96-well plates, 1×10 3 Cells / well; set at 37°C, 5% CO 2 Cultivate in an incubator to make the cells adhere to the wall; add wild-type chimera SDF-1WT-IgG1HCHR-Decorin and mutant chimera SDF-54 / R-IgG1HCHR-Decorin proteins at a predetermined final concentration, place at 37°C, 5% CO 2 Incubator, start timing culture, take out the 96-well plate at 0 hr (0 o'clock when the drug is added), 24 hr, 48 hr, 72 hr, 96 hr, 120 hr, 144 hr and other time points for routine MTT detection, and finally perform data statistics , with the OD (490nm) value on the vertical axis and the treatment time on the horizontal axis, depicting the inhibitory effect of SDF-1WT-IgG1HCHR-Decorin and SDF-54 / R-IgG1HCHR-Decorin on the growth of breast cancer cell MDA-MB-231.

[0099] 2. SDF-54 / R-IgG1HCHR-Decorin induces migration activity of MDA-...

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PUM

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Abstract

The invention provides preparing method of the dual targets effect gene mosaic recombinant and application. The method comprises the following steps: using the SDF-54 / R / pET-30a(+) as match plate, augmenting the SDF-54 / R with specificity primer, entering the SDF-54 / R into T-A clone carrier, then in the 3'-SDF-54 / R joining the butt hinge coded sequence of human IgG1 heavy chain antibody and Decorin mature peptide coded sequence, and constructing the SDF-54 / R-IgG1HCHR-Decorin / pMD18-T recombination mosaic. The SDF-54 / R-IgG1HCHR-Decorin keeps the affinity of CXCR4 and losses the intrinsic activity, and suppresses the chemotaxis and transfer activity of wild-type SDF-1alpha to breast cancer cell and the growth of breast cancer cell. It can be used as CXCR4 antagonist, at the same time suppressing the growth and transfer of CXCR4 positive tumour cell. So it possesses the dual targets effect.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a chimeric recombinant with dual target effect genes capable of inhibiting the metastasis of breast cancer and the growth of breast cancer, its construction method and application. Background technique [0002] The metastasis of malignant tumors is not random migration. Different types of cancer cells migrate to specific organs according to their inherent migration paths. There are many theories trying to explain this phenomenon. The "soil theory" assumes that different organs provide a special microenvironment suitable for the growth of specific cancer cells; the "homing theory" believes that different organs have the ability to recognize, attract or capture specific cancer cells. However, there has been a lack of sufficient convincing evidence for the internal mechanism of this phenomenon over the years (Phlip M. Murphy Chemokines and the molecular basis of cancer meta...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/09A61K48/00A61K38/16A61P35/00
Inventor 蔡绍晖杜军谭毅蔡绍皙马伟峰陈宏远郭自刚
Owner JINAN UNIVERSITY