CN101429525B

CN101429525B - Antineoplastic invasion transfer function of snake venom metalloprotease inhibitors BJ46a and uses thereof

Info

Publication number: CN101429525B
Application number: CN 200710009950
Authority: CN
Inventors: 林建银; 林旭; 徐剑文
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2007-12-07
Filing date: 2007-12-07
Publication date: 2011-09-07
Anticipated expiration: 2027-12-07
Also published as: CN101429525A

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

Antineoplastic invasion transfer function of snake venom metalloprotease inhibitors BJ 46 and application

Technical field

The present invention relates to field of medical biotechnology, a kind of specifically antineoplastic invasion transfer function of snake venom metalloprotease inhibitors BJ 46 and application.

Background technology

The sickness rate of malignant tumour has risen since the seventies in 20th century in the world year by year, and is in second in the dead cause of disease of China.There is the new cancer stricken of 1,800,000 people present every year in China, and 1,500,000 people die from cancer.Malignant tumour brings spirit and sensual dual misery not only for patient and relatives thereof, also the limited health resources of China is caused huge pressure.Control and treatment malignant tumour are one of great public health problems of facing of the Chinese government.

The ability that the propagation of tumor tissue is out of control, the prosoplasia of oncocyte, oncocyte have Invasion and Metastasis is the most basic biological characteristics of malignant tumour, and Invasion and Metastasis is the major cause that malignant tumour threatens patient health and even life.The Invasion and Metastasis of malignant tumour be a complexity process, be broadly divided into following step: the capillary hemangioma bolt forms, tumour cell penetrates basilar membrane, tumour cell arrives secondary position, secondary tumor growth etc.Wherein tumour cell break through extracellular matrix (extracellular matrix, barrier structure ECM) is most important, relate to the most important enzyme of this process system and be matrix metalloproteinase (matrixmetalloproteinase, MMPs).

MMPs is the hydrolysising protease that a class contains calcium and zine ion, and the whole ECM except that polysaccharide of almost degrading have found kind more than 20 at present.In healthy tissues, the level of MMPs is lower and actively be subjected to its natural inhibition-tissue inhibitor of metalloproteinase (tissue inhibitor ofmetalloproteinases, inhibition TIMPs).TIMPs is divided into two functional zone, and the cysteine residues of its N end functional zone combines with the zine ion active centre of MMPs, and C end functional zone combine with other positions of MMPs, form the MMP-TIMP complex body with 1: 1 ratio, thereby blocking-up MMP combines with substrate.In the aggressive malignant tumour, MMPs is high level expression, makes the ECM excessive degradation, organizes basilar membrane and then invasion and attack, transfer to create precondition for tumour cell passes.In addition, MMPs also participates in tumor-blood-vessel growth.Yamamoto etc. have studied 30 routine primary hepatocarcinoma, find that MMP-2 expresses rising and soaks into hepatic vein, shift in the liver and the postoperative recurrence rate is relevant first, show liver cancer cell excretory MMP-2 in the degradation of cell epimatrix, promote to play a crucial role in growth of tumor and the Invasion and Metastasis; Nomura adopts multiple survey inspection means such as immunohistochemical methods, gelatin zymogram, finds that MMP-2 mainly expresses in the development of gastric cancer phase, and the activation of MMP-2 precursor is the key link of stomach cancer cell diffusion.In addition, in cases such as lung cancer, knot-rectum cancer, mammary cancer dissimilar MMPs high expression levels appears all.And the expression of MMPs inhibition TIMPs increases, and the tumor cell invasion transfer ability is weakened.The MMP knock-out mice prompting that developed recently gets up: lack then vasculogenesis minimizing of MMP-2, tumour evolution slows down.Show that after deliberation using the immunohistochemical methods method detects the MMP-9 of stomach cancer cell and the expression of TIMP-1, finds that MMPs and TIMPs imbalance of expression and cancer of the stomach Invasion and Metastasis are in close relations.In a word, to relate to the Invasion and Metastasis of tumour be one than mature theory to MMPs.Therefore, seek novel, potent inhibitors of metalloproteinase and become one of important subject of antineoplastic invasion transfer.

BJ46a (46-kDa isoform a from B.jararaca) is the metalloid protease inhibitor from America spearhead Pallas pit viper (Bothropsjararaca) serum, belongs to Pp63 glycophosphoproteins (fetuin) family of cystatin superfamily.The research of Pp63 glycophosphoproteins continues over half a century, and is still in the ascendant so far.As the branch of cystatin superfamily cystatin, Pp63 glycophosphoproteins has the various biological effect: play an important role in bone remodeling, tire brain development, and can keep in-vitro cell growth; At tumor cell surface, Pp63 glycophosphoproteins and MMPs combination may be in close relations with generation, the development of tumour.The BJ46a molecular weight is 46kDa, is acid glycoprotein, has a double end cystatin structural domain; Be to contain 19 amino acid whose signal peptides before 322 amino acid, total length has 4 N-glycosylation sites of inferring.Experiment shows, BJ46a is by the formation and the metalloprotease interaction of non-covalent complex, can effectively suppress snake venom metalloproteinase (snake venom metalloproteinases, SVMPs) astrolysinc (I class SVMPs) and jararhagin (III class SVMPs) proteolytic activity, inhibiting rate is respectively 100% and 91%.

According to number of ligands, classification and the distribution situation of zinc ion coordination, SVMPs is considered to belong to Metzincins (zinc dependence metalloprotease) family together with MMPs, overall space structure is closely similar.BJ46a can effectively suppress the SVMPs proteolytic activity, and action site is at the metalloprotease structural domain, and it also may be by combining with MMPs, thereby suppresses the Invasion and Metastasis of tumour.

Show that from the pertinent data retrieval that comprises Chinese patent prevention and treatment aspect that snake venom metalloprotease inhibitors BJ46a has the antineoplastic invasion transferance and is applied to tumour are not seen bibliographical information as yet.

Summary of the invention

In order to overcome the deficiencies in the prior art, the objective of the invention is to provide a kind of snake venom metalloprotease inhibitors BJ46a that has the antineoplastic invasion transferance and be applied to prevent and treat the Invasion and Metastasis of tumour.

The technical solution adopted for the present invention to solve the technical problems is: a kind of preparation method of antineoplastic invasion transfer function of snake venom metalloprotease inhibitors BJ 46 and application is characterized in that:

1.BJ46a the structure of rhabdovirus expression vector

According to sequence A F294836 among the GenBank, design and corresponding 20 the complementary oligonucleotide of synthetic, and 10 pairs of PCR primers, make it to be spliced into the BJ46a gene by slow annealing PCR method, directed cloning is to carrier pET-42a (+) behind SacI and XhoI double digestion, and pcr amplification, enzyme are cut and sequencing analysis; With the fixed-point mutation method site that rights a wrong one by one, obtain to contain pET-42a (+)/BJ46a plasmid of correct goal gene;

On the basis of synthetic BJ46a gene, be inserted among the MCS of baculovirus transposon vector pFastBac HT C, transformed into escherichia coli DH5 α-T1, cut evaluation with LB/AP plate screening, pcr amplification and enzyme and obtain positive recombinant, extract pFastBac HT C-BJ46a recombinant chou and further transform the DH10Bac competent cell, screen in vain by blue behind the 48h, the single white colony line of picking, the gained bacterium colony is hickie, thereby makes up baculovirus reorganization transposon vector, recombinant shuttle vector;

2. reorganization expression, the purifying of BJ46a albumen in the Sf9 insect cell

Utilize known baculovirus expression system to produce albumen, mix with recon Bacmid-BJ46a with the Cellfectin liposome, transfection Sf 9 insect cell obtains P1 virus; P1 virus infected insect cell again obtains the P2 virus of higher titre, and then the P3 virus that increases; With the recombinate shape virus infection Sf9 of higher titre, Western blot shows reorganization BJ46a protein expression, and optimum expression time is for infecting back 96h; With titre 5 * 10 ⁷The recombinant virus P3 of pfu/ml infects the Sf9 cell, and the MOI value is 10pfu/cell, 96h collecting cell after infection; Use ProBond ^TMPurification system carries out purifying to the fusion rotein of extensive expression, the SDS-PAGE electrophoretic separation, and coomassie brilliant blue staining and Western-blot analyze the clear band of visible recombination fusion protein;

3. reorganization BJ46a biological activity of albumen

MMP (collagenase) hydrolysis substrate gelatin produces fluorescent substance, during 515nm tangible absorbance value is arranged at excitation wavelength EX 495nm, emission wavelength EM, and substrate gelatin does not have absorbance value at the respective wavelength place; Matrix metalloproteinase Clostridium collagenase final concentration is respectively 0.05U/mL, 0.2U/mL, 0.4U/mL, room temperature, detect the OD value in different time points with spectrophotofluorometer, the result shows: 0.2U/mL, 0.4U/mL Clostridium collagenase fluorescent value within a short period of time rise obviously;

Use EnzChek Gelatinase/Collagenase Assay Kit to detect reorganization BJ46a albumen and suppress the MMPs activity; Reach the purpose that detects enzymic activity by the growing amount that detects hydrolysate; Concentration with adding inhibitor B J46a is X-coordinate, matrix metalloproteinase residual activity % is an ordinate zou, draws the inhibition activity curve of recombinant protein BJ46a to MMPs, shows: along with increasing of recombinant protein concentration, MMPs's is active on a declining curve, and BJ46a suppresses active IC to MMPs ₅₀Value is 0.12mg/ml;

4. the detection of the outer antineoplastic invasion transferance of reorganization BJ46a proteoplast

Use the Transwell cell and analyze the external basilar membrane ability of passing through of B16, reorganization BJ46a albumen treatment group B16 cell count is 5.93 ± 0.66, is lower than 39 ± 2.79 of control group, P＜0.01, inhibiting rate 84.8% and blank group 41.4 ± 3.37, P＜0.01;

5. use B16 cell experiment lung metastasis model and analyze Invasion and Metastasis ability in the tumour cell body

Different concns reorganization BJ46a albumen treatment group lung metastatic tumor number is 1.1 ± 0.83,0.9 ± 0.7, is lower than 6.3 ± 3.00 of control group, P＜0.001 and blank group 10.7 ± 5.73, P＜0.001;

6. make up the screening of pcDNA3.1HisC-BJ46a carrier and stable transfection B16 clone thereof

With conventional Protocols in Molecular Biology BJ46a is cloned in the pcDNA3.1HisC plasmid, makes up the pcDNA3.1HisC-BJ46a carrier; Fugene 6 method transfection B16 cells, through G418 screening resistant cell clone, RT-PCR identifies that its BJ46a mRNA expresses;

7.B16/pcDNA3.1HisC-BJ46a the external invasive ability analysis of stable transfected cells strain

Adopt Transwell cell Invasion and Metastasis model result to show: the external invasive ability of B16/pcDNA3.1HisC-BJ46a stable transfected cells strain obviously reduces, it is worn the theca cell number and is lower than B16/pcDNA3.1HisC empty carrier group and blank B16 groups of cells (P＜0.01), inhibiting rate is 59.2%, and its in-vitro multiplication, adhesion, motor capacity are not seen obvious change;

8.B16/pcDNA3.1HisC-BJ46a the mouse experiment lung metastasis model of stable transfected cells

Adopt the C57BL/6 mouse to set up the experimental lung metastasis model through tail vein injection B16/pcDNA3.1HisC-BJ46a cell, B16/pcDNA3.1HisC cell, B16 cell, inject back 20 days with cervical vertebra dislocation method execution mouse, the lung rate of transform is 100%, but B16/pcDNA3.1HisC-BJ46a organizes its lung's metastasis number, histology and ultrastructure all is better than control group, thereby shows that the BJ46a stable transfection can suppress the B16 cell invasion and shift.

The invention has the beneficial effects as follows: because the present invention according to the BJ46a gene order (AF294836) among the GenBank, designs and the full gene of synthetic BJ46a.Be cloned into rhabdovirus expression vector, have the reorganization BJ46a albumen that suppresses matrix metal proteinase activity in the production of Sf9 insect cell, and prove that by mouse experiment lung shift experiment in external invasive model test and the body it has melanoma cell B16 Invasion and Metastasis function.Therefore, the present invention can be used as the protein drug that the genetically engineered production application is shifted in prevention and treatment tumor invasion.

The present invention makes up the pcDNA3.1HisC-BJ46a carrier for expression of eukaryon, and the applying gene rotaring dyeing technology is set up the B16/pcDNA3.1HisC-BJ46a cell strain of stable transfection, proves once more that by experiment in vivo and vitro BJ46a has obvious antineoplastic invasion transferance.Therefore, the present invention also can be used as the Invasion and Metastasis that gene therapy is applied to prevent and treat tumour.

By the synthetic BJ46a gene of genetically engineered, has the potential that suppresses the transfer of B16 cell invasion from albumen and gene level proof BJ46a first.Therefore,, can prepare reorganization BJ46a albumen on the one hand in a large number, as the Invasion and Metastasis of engineered protein medicinal application in prevention and treatment tumour by biotechnology; On the other hand, make up corresponding BJ46a expression vector or virus, be applied to prevent and treat the Invasion and Metastasis of tumour as gene therapy.This shows that BJ46a will have great application prospect and economic results in society aspect the Invasion and Metastasis of prevention and treatment tumour.

Description of drawings

Fig. 1: BJ46a gene amplification synoptic diagram.

Fig. 2: the polymerization kinetics curves of matrix metalloproteinase hydrolysis substrate gelatin (X-coordinate is the time: minute; Ordinate zou is an absorbancy).

Fig. 3: reorganization BJ46a albumen suppresses matrix metal proteinase activity analysis (X-coordinate is reorganization BJ46a protein concentration mg/ml, and ordinate zou is matrix metalloproteinase residual activity %).

Fig. 4: the pre-treatment of reorganization BJ46a albumen (is followed successively by reorganization BJ46a protein groups, foreign protein control group, blank group to the influence of B16 cell invasion ability in the X-coordinate; Ordinate zou is the cell count of invasion and attack).

Fig. 5: inoculation reorganization BJ46a albumen pre-treatment B16 cell C57BL/6 mouse lungs metastatic tumor kitchen ranges (A is the blank group, and B is the foreign protein control group, and C is reorganization BJ46a protein groups).

Fig. 6: the BJ46a stable transfection is to the influence (A is the B16/pcDNA3.1HisC-BJ46a group, and B is the B16/pcDNA3.1HisC group, and C is untransfected B16 group haematoxylin dyeing * 200) of B16 cell invasion ability.

Fig. 7: inoculation B16 cell strain C57BL/6 mouse lung metastatic tumor histological observation (A is the B16 group, and B is the B16/pcDNA3.1HisC group, and C is B16/pcDNA3.1HisC-BJ46a group HE dyeing * 100).

Embodiment

Below the invention will be further described by specific embodiment

Embodiment 1:

The BJ46a gene is synthetic

According to the BJ46a gene order (AF294836) of searching among the GenBank it is divided into 20 fragments, and 10 pairs of primers of respective design, synthesizing and purifying.

20 fragment sequences are respectively:

1：5’-ATG?AAT?TCC?CTG?GTA?GCT?CTC?GTG?CTC?CTG?GGT?CAG?ATT?ATA?GGA?TCTACG?CTT?AGC?TCT?CAA?GTG?AGG?G-3’

2：5’-ACC?TGT?ATC?TGT?CCA?CTC?TTT?AGC?GTC?TTT?CTC?GTC?GCA?TTC?TAA?ATCCCC?CCT?CAC?TTG?AGA?GCT?AAG?C-3’

3：5’-GAG?TGG?ACA?GAT?ACA?GGT?GTG?CGC?TAC?ATC?AAC?GAG?CAT?AAA?CTA?CATGGA?TAC?AAA?TAT?GCC?CTC?AAT?GTA?A-3’

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’

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’

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’

7：5’-ATG?CTG?TTG?AAA?TGG?ACT?GTG?ATG?TCA?AGA?TAA?TGT?TTA?ATG?TTG?ATACTT?TCA?AAG?AAG?ATG?TTT?TTG?C-3’

8：5’-GAC?AAT?TTC?GCC?GCA?CGT?TTT?CCA?CAG?AAT?CTG?GAG?TGG?AGT?GGC?ATTTTG?CAA?AAA?CAT?CTT?CTT?TGA?A-3’

9：5’-AAA?CGT?GCG?GCG?AAA?TTG?TCC?TAA?ATG?TCC?AAT?TCT?GTT?GCC?TTC?GAATAA?CCC?TCA?GGT?GGT?AGA?CTC?T-3’

10：5’-TAA?ACG?TGG?TCG?GAA?AGT?TTT?TCA?TTG?TGT?TTA?TTA?AGC?ACA?TAT?TCAACA?GAG?TCT?ACC?ACC?TGA?GGG?T-3’

11：5’-AAA?CTT?TCC?GAC?CAC?GTT?TAC?GAA?GTT?CTT?GAG?ATT?TCA?AGA?GGG?CAGCAC?AAA?TAT?GAG?CCT?GAA?GCT?T-3’

12：5’-GAG?TTC?CTG?AGC?AGT?GCA?GTT?AAC?CTC?CAC?AAT?AGC?AAA?CTC?CAC?ATAGTA?AGC?TTC?AGG?CTC?ATA?TTT?G-3’

13：5’-ACT?GCA?CTG?CTC?AGG?AAC?TCC?ACG?ATG?ACC?ATC?ATC?ACT?GCC?ATC?CTAATA?CAG?CAG?GAG?AAG?ACC?ATA?T-3’

14：5’-GTT?TTT?CCA?GGC?TAG?CAT?GCG?ACC?TGA?AAA?CAG?TTG?CTC?TGC?AGA?ATCCAA?TAT?GGT?CTT?CTC?CTG?CTG?T-3’

15：5’-GCA?TGC?TAG?CCT?GGA?AAA?ACC?TAA?AGA?TGA?ACA?GTT?TGA?GTC?GGA?CTGTGT?CAT?CCT?TCA?TGT?CAA?GGA?G-3’

16：5’-ATA?CTG?TCT?TTT?TCA?ACG?TGC?TGC?TGA?ATC?AAA?TGG?GAG?TGA?GCG?TGTCCC?TCC?TTG?ACA?TGA?AGG?ATG?A-3’

17：5’-CAC?GTT?GAA?AAA?GAC?AGT?ATT?TCC?CCA?GAA?CAC?AAC?AAT?ACT?GCC?CTCAAC?TTC?GTC?CAT?CCA?CAC?AAT?G-3’

18：5’-AAC?TGG?GAC?TTC?CGC?CAA?ATG?TTC?ATG?AGA?CTC?GTG?TGA?GGT?GCT?TGTATC?ATT?GTG?TGG?ATG?GAC?GAA?G-3’

19：5’-ATT?TGG?CGG?AAG?TCC?CAG?TTG?CTT?TTG?TCA?AAA?AAG?AAC?TCC?CCA?AAGATA?TAT?CAG?ATC?GTC?ACA?CAA?C-3’

20：5’-CTA?CAG?CTC?GAA?GTG?ATG?TAC?TTT?TCC?TGG?ACA?ACC?TTT?CAC?AGG?GGTTGT?GTG?ACG?ATC?TGA?TAT-3’

10 pairs of primer sequences are respectively:

12F：5’-ATG?AAT?TCC?CTG?GTA?GCT?C-3’

12R：5’-ACC?TGT?ATC?TGT?CCA?CTC-3’

34F：5’-GAG?TGG?ACA?GAT?ACA?GG-3’

34R：5’-TTA?AGG?AAG?ACT?GCC?ACC?C-3’

56F：5’-TGG?GTG?GCA?GTC?TTC?CTT-3’

56R：5’-ACA?GTC?CAT?TTC?AAC?AGC-3’

78F：5’-ATG?CTG?TTG?AAA?TGG?ACT?G-3’

78R：5’-GAC?AAT?TTC?GCC?GCA?CGT?TTT?C-3’

910F：5’-AAA?CGT?GCG?GCG?AAA?TTG-3’

910R：5’-TAA?ACG?TGG?TCG?GAA?AG-3’

1112F：5’-AAA?CTT?TCC?GAC?CAC?GTT-3’

1112R：5’-GAG?TTC?CTG?AGC?AGT?GCA?G-3’

1314F：5’-ACT?GCA?CTG?CTC?AGG?AAC?TC-3’

1314R：5’-GTT?TTT?CCA?GGC?TAG?CAT?GCG-3’

1516F：5’-GCA?TGC?TAG?CCT?GGA?AAA?AC-3’

1516R：5’-ATA?CTG?TCT?TTT?TCA?ACG?TG-3’

1718F：5’-CAC?GTT?GAA?AAA?GAC?AG-3’

1718R：5’-AAC?TGG?GAC?TTC?CGC?CAA?ATG-3’

1920F：5’-ATT?TGG?CGG?AAG?TCC?CAG?TTG-3’

1920R：5’-CTA?CAG?CTC?GAA?GTG?ATG?TAC-3’

Through agarose gel electrophoresis, observations under the ultraviolet lamp: 20 fragments obtain 10 dna fragmentations of 12,34,56,78,910,1112,1314,1516,1718,1920 about 120bp respectively after slowly annealing PCR splices in twos; 12,34,910,1112,1314,1516,1718,1,920 4 fragments that further are spliced into 14,912,1316,1720 about 220bp; 14,56 be spliced into 16,78,912 and be spliced into 712, be i.e. 2 of about 320bp fragments; 16,712 be spliced into 112,1316,1720 of about 620bp and be spliced into 1320 of about 420bp; 112,1320 be spliced into 120 of about 1020bp, promptly complete BJ46a gene.These segmental sizes are consistent with expected results.The synthetic synoptic diagram of BJ46a gene is seen Fig. 1.

Embodiment 2:

By GeneTailor locus specificity abruptly-changing system error recovery base

The synthetic back of BJ46a sequencing shows and snake class BJ46a gene order basically identical, but mistake appears in 6 sites.According to sequencing result with the method for the rite-directed mutagenesis site that rights a wrong one by one.Each site mutation all comprises three steps: at first use site-specific methylase that target DNA is methylated; There is the eclipsed primer to carry out jump reaction by two then, wherein a primer band purpose mutational site; The product that will suddenly change at last is transformed into bacillus coli DH 5 alpha-T1 of mcrBC+, chooses the clone, identifies by SacI and XhoI double digestion, and has finished desired sudden change through this site of order-checking confirmation.Extraction contains mutator gene plasmid, and overline three steps are finished another site mutation, obtain correct goal gene at last.

Embodiment 3:

The structure of BJ46a rhabdovirus expression vector

BJ46a is inserted among the MCS of baculovirus transposon vector pFastBac HT C behind SacI and XhoI site double digestion, transformed into escherichia coli DH5 α-T1, with LB/AP plate screening positive recombinant, pcr amplification, enzyme are cut evaluation, extract pFastBac HT C-BJ46a recombinant chou and further transform the DH10Bac competent cell, screen in vain by blue behind the 48h, the single white colony line of picking, confirm that the gained bacterium colony is hickie, choose clone PCR and identify.The result shows and successfully makes up baculovirus reorganization transposon vector, recombinant shuttle vector.The high purity plasmid Bacmid-BJ46a recombinant chou that the extracting cell transfecting is used.

Embodiment 4:

Expression, purifying, the evaluation of reorganization BJ46a albumen in the Sf9 insect cell

Mix with the Bacmid-BJ46a recombinant chou with the Cellfectin liposome, transfection Sf9 cell is collected and is contained recombinant baculovirus particulate P1 virus; Get transfection supernatant P1 and infect the P2 virus that the Sf9 cell obtains higher titre with MOI 0.1, and then amplification P3 virus.

Be in the Sf9 cell of logarithmic phase, calculate cell survival rate with trypan blue dyeing, select for use survival rate more than or equal to 97% cell inoculation in 12 orifice plates, density is 1 * 10 ⁶Cells/well.The adherent 1h of room temperature carries out recombinant virus Bacmid-BJ46a inoculation with MOI 10, infects the Sf9 cell.Respectively 24,48,72,96,120h inhales and abandons cell culture fluid, PBS gives a baby a bath on the third day after its birth time, the exhaustion raffinate.Every porocyte adds 1 * sample-loading buffer, and (final concentration is 0.08M Tris-HCl, 2%SDS, 10% glycerine, 5% beta-mercaptoethanol, 2% bromjophenol blue) 200 μ l, 4 ℃ of lysing cell 30min, lysate is transferred in the centrifuge tube, and boiling water bath 10min makes the thorough sex change of albumen, 4 ℃ of centrifugal 10min of 12000g remove chip, and-20 ℃ of preservations are standby.With the SDS-PAGE electrophoretic separation, coomassie brilliant blue staining and Western-blot analyze expression product, the result shows: infect recombinant virus 48,72,96,120h visible obviously band---recombination fusion protein between 48.8-64.2kDa, infect cell and normal Sf9 cell that recombinant virus Bacmid does not take place and there is no this band.The optimum expressing fusion protein time is for infecting back 96h.

Large scale culturing Sf9 cell, amplicon virus.With high titre recombinant virus infection Sf9 cell, the MOI value is 10pfu/cell, at the optimum expression time point---infect back 96h collecting cell.Add 6ml Native Binding damping fluid re-suspended cell, through twice freeze-thaw cycle, 1ml one-shot injector suction 4 times, the centrifugal 10min of 4 ℃ of 12000g of lysate removes chip, uses ProBond ^TMPurification system carries out purifying to fusion rotein.The capable SDS-PAGE electrophoretic separation of purified product, coomassie brilliant blue staining and Western-blot analyze.The Western-blot concrete grammar is: after electrophoresis finishes, albumen on the glue is changeed film instrument (250mA by electricity, 3h) be transferred on the nitrocellulose filter, sealing is spent the night among the TBS of 0.3%BSA, order adds one and anti-is the anti-BJ46a antibody of rabbit, two anti-are alkali phosphatase enzyme mark goat anti-rabbit igg, NBT/BCIP colour developing.The result shows successful purification of Recombinant BJ46a albumen.In reorganization BJ46a albumen purification process, with the Sf9 cell of similarity condition purifying infection empty carrier Bacmid, the foreign protein that obtains is as the contrast of subsequent recombination BJ46a protein series functional experiment.

Embodiment 5:

The proteic biologic activity analysis of reorganization BJ46a

See also Fig. 2, Fig. 3, MMP (collagenase) hydrolysis substrate gelatin produces fluorescent substance, during 515nm tangible absorbance value is arranged at excitation wavelength EX 495nm, emission wavelength EM, and substrate gelatin does not almost have absorbance value at the respective wavelength place.Matrix metalloproteinase Clostridium collagenase final concentration is respectively 0.05U/mL, 0.2U/mL, 0.4U/mL, room temperature, detect the OD value in different time points with spectrophotofluorometer, the result shows: 0.2U/mL, the rising of 0.4U/mL Clostridium collagenase fluorescent value within a short period of time be (Fig. 2) obviously.

Use EnzChek Gelatinase/Collagenase Assay Kit to detect reorganization BJ46a albumen and suppress the MMPs activity.Can reach the purpose that detects enzymic activity by the growing amount that detects hydrolysate.Concentration with adding inhibitor B J46a is X-coordinate, and matrix metalloproteinase residual activity % is an ordinate zou, draws the inhibition activity curve of recombinant protein BJ46a to MMPs.The result shows: along with increasing of recombinant protein concentration, MMPs's is active on a declining curve, and BJ46a suppresses active IC to MMPs ₅₀Value is 0.12mg/ml (Fig. 3).

Embodiment 6:

The B16 cells in vitro motion that reorganization BJ46a albumen is handled and the detection of invasive ability

See also Fig. 4, adopt Transwell cell (Transwell chamber) analytical procedure evaluation reorganization BJ46a albumen to handle back B16 cells in vitro motor capacity and invasive ability.

At first Transwell (Corning Costar 3422 types, USA) be inverted, it is air-dry to drip 10 μ l Fibronect in (0.5mg/ml) room temperature 1h at Transwell invasion and attack cell polycarbonate membrane lower surface, the inboard 20 μ l Matrigel (1: 8 diluent of 9.1mg/ml Matrigel) that add of film, 37 ℃ of 1h make Matrigel form gel.The B16 cell that is in logarithmic phase is through trysinization, and the PBS washing is resuspended in the 0.1%BSA-serum-free RPMI-1640, is mixed with 2 * 10 ⁶Individual cell/ml, add reorganization BJ46a albumen, making its final concentration is 0.2mg/ml, with foreign protein treatments B 16 cells is control group, simple B16 cell is the blank group, adds chamber on the invasion and attack cell behind 37 ℃ of 5%CO2 incubator incubation 2h, and following chamber adds 600 μ l and contains 10% foetal calf serum, 1640 liquid, put 37 ℃, 5%CO ₂Cultivate 48h.Take out chamber liquid on the cell reject, go up cell and the Matrigel that the chamber face is not worn film to the greatest extent with the cotton swab wiping, the fixing 5min of methyl alcohol under the room temperature, haematoxylin dyeing, 400 times of light microscopics are counted the invasion and attack cell count in 5 visuals field down, get its mean value, represent the invasion by tumor cells ability with the relative number of invasion and attack cell.Every group of experiment repeats 3 times.

Do not add Matrigel in the polycarbonate membrane inboard during cell chemotaxis motion analysis, 37 ℃, 5%CO ₂Cultivate 18h, all the other operation stepss and method of counting are tested with external invasion and attack.

The result is shown in Fig. 4 and table 1, it is 5.93 ± 0.66 that the cell count of rebuilding basilar membrane is passed through in reorganization BJ46a albumen treatment group B16 invasion and attack, be starkly lower than control group (39 ± 2.79, P＜0.01, inhibiting rate 84.8%) and blank group (41.4 ± 3.37, P＜0.01), difference has remarkable statistical significance.This prompting BJ46a albumen has the potential that suppresses the invasion and attack of B16 cells in vitro, but to the not influence of B16 cells in vitro motor capacity.

Table 1 is through pretreated B16 cells in vitro migration of reorganization BJ46a albumen and invasive ability (n=3)

* a.b, a.c compare, and #b.c relatively

Embodiment 7:

Reorganization BJ46a albumen pre-treatment B16 cell mouse experimental lung metastasis

See also Fig. 5,40 of C57BL/6 mouse, heavy (16-18g) is divided into 4 groups, 10 every group at random by animal body.The B16 cell of taking the logarithm respectively vegetative period, trysinization and centrifugation cell, PBS liquid cleans cell 2 times, and with PBS liquid re-suspended cell, adjusting concentration is 1 * 10 ⁶The single cell suspension of individual/ml, the reorganization BJ46a albumen of adding different concns is set up PBS blank group, foreign protein control group simultaneously.In C57BL/6 mouse tail vein injection 200 μ l tumor cell suspensions.Inject and put to death mouse in back 20 days, get lung tissue.Counting lung metastasis tubercle number.Tissue is fixed through neutral formalin, paraffin embedding, section, and HE dyeing, opticmicroscope is observed its pathological change down.

The result is shown in Fig. 5 and table 2: as seen control group mice lung is dispersed in the metastases kitchen range in a large number, and the transfer of diffusivity thoracic cavity occurs; Most lungs metastasis merges in the form of sheets under the light microscopic, and the oncocyte volume is big in the metastasis, and cell shape is different, and nuclear atypia is obvious, visible volume melanin granule.Reorganization BJ46a albumen treatment group, lungs metastatic tumor kitchen range is little, and knurl tubercle number obviously reduces, and compares with control group, and difference has remarkable statistical significance (P＜0.001), illustrates that reorganization BJ46a albumen can suppress the experimental lung metastasis of B16 cell.The knurl tubercle number that the reorganization BJ46a protein groups of final concentration 0.3mg/ml forms is slightly less than the 0.2mg/ml group, but the two differences not statistically significant.

Table 2 inoculation BJ46a albumen pre-treatment B16 cell C57BL/6 mouse lung metastatic tumor kitchen range number (n=10)

Group	Numbers?of?Metastatic lesions?in?lung	The P value
			BJ46a(0.2mg/ml) ^a BJ46a(0.3mg/ml) ^b control ^c blank?control ^d	1.1±0.83 0.9±0.7 6.3±3.00 10.7±5.73	P*＞0.001 P#＞0.05?

* a.c, a.d, b.c, b.d are relatively

#a.b, c.d are relatively

Embodiment 8:

The structure of recombinant plasmid pcDNA3.1 HisC-BJ46a

Correct BJ46a gene with step 2 is that template is carried out pcr amplification, upstream primer is 5 '- GGGGTACCATCTCAAGTGAGGGGGGATTTAG-3 ' (underscore is the KpnI restriction enzyme site), downstream primer be 5 '- CCGCTCGAGCTACAGCTCG AAGTGATG-3 ' (underscore is the XhoI restriction enzyme site).BJ46a gene forward behind double digestion of introducing KpnI and XhoI restriction enzyme site inserts pcDNA3.1HisC, connect product transformed into escherichia coli Top10, place 37 ℃ of incubator 16h, two clones of picking at random on penbritin (concentration is 100 μ g/ml) flat board, shake bacterium and spend the night, extract plasmid DNA.Sequencing result shows that BJ46a DNA inserts fragment sequence and reading frame is entirely true, the carrier called after pcDNA3.1HisC-BJ46a of structure.

Embodiment 9:

The screening of pcDNA3.1HisC-BJ46a transfection B16 cell and stably transfected cell line

Transfection the day before yesterday is with the B16 cell trysinization of logarithmic phase, with 3 * 10 ⁵Individual/hole is inoculated in 6 orifice plates, adds the RPMI-1640 nutrient solution that contains 10% foetal calf serum, to transfection remittance sheet on the same day 50%～80%.Transfection reagent Fugene 6 gets 3ul, and RPMI-1640 is diluted to 100ul with serum free medium, and mixing is hatched 5min.Directly add DNA at 3: 2 according to transfection reagent: DNA (ug) ratio, mixing is also hatched 30min.Dropwise evenly add in 6 orifice plates of culturing cell.Changing the nutrient solution that contains G418 (600 μ g/ml) behind the transfection 24h screens.Behind the 2w, recombinant plasmid group, empty carrier group all have the resistant cell clone to form and all death of simple B16 cell control group.It is some that transfection 3w selects unicellular positive colony, changes the nutrient solution enlarged culturing that contains G418 (300 μ g/ml) again in case spline is gone into gene loses.

Behind 600 μ g/ml G418 resistance screening cloning cells, the RT-PCR method is size test B16/pcDNA3.1HisC-BJ46a cell destination gene expression just, the result shows: the cell clone of 2 BJ46a stable transfections all detects the cDNA fragment about 1kp, and the B16/pcDNA3.1HisC cell of transfection empty carrier does not detect above-mentioned band.

Embodiment 10:

The detection of the external invasive ability of B16 cell strain of BJ46a stable transfection

See also Fig. 6, in Transwell invasion and attack cell (Corning Costar 3422 types, USA) the polycarbonate membrane outside drips 10 μ l FN (0.5mg/ml) and air-dry, inboard 20 μ l Matrigel (the stoste 9.1mg/ml that add of film, dilute by 1: 8 with serum-free RPMI-1640 nutrient solution), air-dry.Indoorly on cell add above-mentioned B16/pcDNA3.1HisC-BJ46a cell, B16/pcDNA3.1HisC cell and the blank B16 cell that 200 μ l are resuspended in 0.1%BSA-serum-free RPMI-1640 respectively and (contain 2 * 10 ⁵Individual cell), following chamber adds the RPMI-1640 nutrient solution that 600 μ l contain 10%FBS, puts 37 ℃, 5%CO ₂Incubator 48h.Take out chamber liquid on the cell reject, wipe with cotton swab and go up the cell that the chamber face is not worn film to the greatest extent, the fixing 5min of methyl alcohol under the room temperature, conventional haematoxylin dyeing, 200 times of light microscopics are counted the invasion and attack cell count in 5 visuals field down, get its mean value, represent the invasion by tumor cells ability with the relative number of invasion and attack cell.Every group of experiment repeats 3 times.

The result is shown in Fig. 6, table 3: B16/ pcDNA3.1HisC-BJ46a cell invasion passes through the reconstruction basilar membrane and obviously is suppressed, attack that the cell count of chamber is 8.53 ± 1.60 to the cell, be starkly lower than empty carrier group (20.93 ± 2.16, P＜0.05, inhibiting rate is 59.2%) and blank group (41.60 ± 3.10, P＜0.01).The transfection of prompting BJ46a stable gene can suppress B16 cells in vitro Invasion Potential, but to the not influence of B16 cells in vitro motor capacity.

Table external migration of 3B16 cell clone and invasive ability (n=3)

* a.b, a.c compare, and #b.c relatively

Embodiment 11:

The mouse experiment lung metastasis model of B16/pcDNA3.1HisC-BJ46a stable transfected cells

See also Fig. 7,30 of C57BL/6 mouse are divided into 3 groups at random by body weight, 10 every group.Three groups of cells of the B16/pcDNA3.1HisC-BJ46a that takes the logarithm respectively vegetative period, B16/pcDNA3.1HisC and B16 are blown and beaten and centrifugation cell repeatedly, and PBS liquid cleans 2 times, and with PBS liquid re-suspended cell, adjusting concentration is 1 * 10 ⁶The single cell suspension of individual/ml is in C57BL/6 mouse tail vein injection 200 μ l tumor cell suspensions.Inject back 20 days cervical vertebra dislocation methods and put to death mouse, get lung tissue.Counting lung metastasis tubercle number.Tissue is fixed through neutral formalin, paraffin embedding, section, and HE dyeing, opticmicroscope is observed its pathological change down.In addition, choose the knurl tubercle, fixing before 3% glutaraldehyde-1.5% Paraformaldehyde 96, fixing behind 1% osmic acid-1.5% yellow prussiate of potash, alcohol-acetone dehydration, Resins, epoxy 618 embedding medium embeddings; Ultrathin section(ing), acetic acid uranium, lead citrate dyeing, the Hu-12A of Hitachi type transmission electron microscope observing, photography.

The result shows: three groups of cell C57BL/6 mouse lung rates of transform are 100%, but its lung's metastasis number, histology all have different with ultrastructure.As seen B16/pcDNA3.1HisC groups of cells, blank B16 groups of cells mouse lung are dispersed in the metastases kitchen range in a large number, and the transfer of diffusivity thoracic cavity occurs.B16/pcDNA3.1HisC-BJ46a groups of cells mouse lungs metastatic tumor kitchen range number obviously is less than empty carrier group and blank group, and difference has statistical significance (table 4).Blank B16 groups of cells, B16/pcDNA3.1HisC groups of cells lung metastasis merge in the form of sheets under the light microscopic, form huge knurl kitchen range, and the knurl bolt is arranged in the blood vessel; The oncocyte volume is big in the metastasis, and cell shape is different, and nuclear atypia is obvious, the visible black color crude granule; The B16 cell stretches out projection in the bigger knurl tubercle, interconnects each other, forms loop network shape structure---vasculogenesis mimicry, in red corpuscle is arranged.Transfection BJ46a organizes knurl tubercle little (Fig. 7), illustrates that the stable gene transfection can suppress the experimental lung metastasis of B16 cell.

The C57BL/6 mouse lungs metastatic tumor kitchen range numbers (n=10) of table 4 inoculation B16/BJ46a cell

Group	Numbers?of?Metastatic lesions?in?lung	The P value
			B16/pcDNA3.1HisC-BJ46a ^a B16/pcDNA3.1HisC ^b B16 ^c	4.7±2.76 8.7±4.94 13.3±4.00	P*＜0.05 P#＜0.01

* a.b comparison, #a.c, b.c are relatively

SEQUENCE?LISTING

＜110〉Medical University Of Fujian

＜120〉antineoplastic invasion transfer function of snake venom metalloprotease inhibitors BJ 46 and application

<140>200710009950.0

<141>2007-12-07

<160>2

<170>PatentIn?version?3.1

<210>1

<211>1026

<212>DNA

＜213〉America spearhead Pallas pit viper (Bothrops jararaca)

<400>1

atg?aat?tcc?ctg?gta?gct?ctc?gtg?ctc?ctg?ggt?cag?att?ata?gga 45

Met?Asn?Ser?Leu?Val?Ala?Leu?Val?Leu?Leu?Gly?Gln?Ile?Ile?Gly

1 5 10 15

tct?acg?ctt?agc?tct?caa?gtg?agg?ggg?gat?tta?gaa?tgc?gac?gag 90

Ser?Thr?Leu?Ser?Ser?Gln?Val?Arg?Gly?Asp?Leu?Glu?Cys?Asp?Glu

20 25 30

aaa?gac?gct?aaa?gag?tgg?aca?gat?aca?ggt?gtg?cgc?tac?atc?aac 135

Lys?Asp?Ala?Lys?Glu?Trp?Thr?Asp?Thr?Gly?Val?Arg?Tyr?Ile?Asn

35 40 45

gag?cat?aaa?cta?cat?gga?tac?aaa?tat?gcc?ctc?aat?gta?att?aag 180

Glu?His?Lys?Leu?His?Gly?Tyr?Lys?Tyr?Ala?Leu?Asn?Val?Ile?Lys

50 55 60

aat?atc?gtt?gtc?gtt?ccc?tgg?gat?ggc?gat?tgg?gtg?gca?gtc?ttc 225

Asn?Ile?Val?Val?Val?Pro?Trp?Asp?Gly?Asp?Trp?Val?Ala?Val?Phe

65 70 75

ctt?aaa?tta?aat?ctt?ctg?gag?aca?gaa?tgt?cat?gtg?ttg?gat?cca 270

Leu?Lys?Leu?Asn?Leu?Leu?Glu?Thr?Glu?Cys?His?Val?Leu?Asp?Pro

80 85 90

act?cct?gtc?aag?aat?tgt?act?gta?agg?cca?cag?cat?aat?cat?gct 315

Thr?Pro?Val?Lys?Asn?Cys?Thr?Val?Arg?Pro?Gln?His?Asn?His?Ala

95 100 105

gtt?gaa?atg?gac?tgt?gat?gtc?aag?ata?atg?ttt?aat?gtt?gat?act 360

Val?Glu?Met?Asp?Cys?Asp?Val?Lys?Ile?Met?Phe?Asn?Val?Asp?Thr

110 115 120

ttc?aaa?gaa?gat?gtt?ttt?gca?aaa?tgc?cac?tcc?act?cca?gat?tct 405

Phe?Lys?Glu?Asp?Val?Phe?Ala?Lys?Cys?His?Ser?Thr?Pro?Asp?Ser

125 130 135

gtg?gaa?aac?gtg?cgg?cga?aat?tgt?cct?aaa?tgt?cca?att?ctg?ttg 450

Val?Glu?Asn?Val?Arg?Arg?Asn?Cys?Pro?Lys?Cys?Pro?Ile?Leu?Leu

140 145 150

cct?tcg?aat?aac?cct?cag?gtg?gta?gac?tct?gtt?gaa?tat?gtg?ctt 495

Pro?Ser?Asn?Asn?Pro?Gln?Val?Val?Asp?Ser?Val?Glu?Tyr?val?Leu

155 160 165

aat?aaa?cac?aat?gaa?aaa?ctt?tcc?gac?cac?gtt?tac?gaa?gtt?ctt 540

Asn?Lys?His?Asn?Glu?Lys?leu?Ser?Asp?His?Val?Tyr?Glu?Val?Leu

170 175 180

gag?att?tca?aga?ggg?cag?cac?aaa?tat?gag?cct?gaa?gct?tac?tat 585

Glu?Ile?Ser?Arg?Gly?Gln?His?Lys?Tyr?Glu?Pro?Glu?Ala?Tyr?Tyr

185 190 195

gtg?gag?ttt?gct?att?gtg?gag?gtt?aac?tgc?act?gct?cag?gaa?ctc 630

Val?Glu?Phe?Ala?Ile?Val?Glu?Val?Asn?Cys?Thr?Ala?Gln?Glu?Leu

200 205 210

cac?gat?gac?cat?cat?cac?tgc?cat?cct?aat?aca?gca?gga?gaa?gac 675

His?Asp?Asp?His?His?His?Cys?His?Pro?Asn?Thr?Ala?Gly?Glu?Asp

215 220 225

cat?att?gga?ttc?tgc?aga?gca?act?gtt?ttc?agg?tcg?cat?gct?agc 720

His?Ile?Gly?Phe?Cys?Arg?Ala?Thr?Val?Phe?Arg?Ser?His?Ala?Ser

230 235 240

ctg?gaa?aaa?cct?aaa?gat?gaa?cag?ttt?gag?tcg?gac?tgt?gtc?atc 765

Leu?Glu?Lys?Pro?Lys?Asp?Glu?Gln?Phe?Glu?Ser?Asp?Cys?Val?Ile

245 250 255

ctt?cat?gtc?aag?gag?gga?cac?gct?cac?tcc?cat?ttg?att?cag?cag 810

Leu?His?Val?Lys?Glu?Gly?His?Ala?His?Ser?His?Leu?Ile?Gln?Gln

260 265 270

cac?gtt?gaa?aaa?gac?agt?att?tcc?cca?gaa?cac?aac?aat?act?gcc 855

His?Val?Glu?Lys?Asp?Ser?Ile?Ser?Pro?Glu?His?Asn?Asn?Thr?Ala

275 280 285

ctc?aac?ttc?gtc?cat?cca?cac?aat?gat?aca?agc?acc?tca?cac?gag 900

Leu?Asn?Phe?Val?His?Pro?His?Asn?Asp?Thr?Ser?Thr?Ser?His?Glu

290 295 300

tct?cat?gaa?cat?ttg?gcg?gaa?gtc?cca?gtt?gct?ttt?gtc?aaa?aaa 945

Ser?His?Glu?His?Leu?Ala?Glu?Val?Pro?Val?Ala?Phe?Val?Lys?Lys

305 310 315

gaa?ctc?ccc?aaa?gat?ata?tca?gat?cgt?cac?aca?acc?cct?gtg?aaa 990

Glu?Leu?Pro?Lys?Asp?Ile?Ser?Asp?Arg?His?Thr?Thr?Pro?Val?Lys

320 325 330

ggt?tgt?cca?gga?aaa?gta?cat?cac?ttc?gag?etg?tag 1026

Gly?Cys?Pro?Gly?Lys?Val?His?His?Phe?Glu?Leu

335 340

<210>2

<211>341

<212>PRT