CN1903373A - Application of expression TRAIL protein colon bacillus for preparing medicine to treat tumor - Google Patents

Abstract

An application of the colibacillus expressing TRAIL protein in preparing the medicines for treating tumor is disclosed. Said colibacillus with tumor target and carrying said TRAIL protein can be located in the tumor tissue to make the TRAIL protein to play its stronger suppression role to tumor.

Description

A kind of purposes of escherichia coli in the preparation anti-tumor medicine of expressing trail protein matter

Technical field

The present invention relates to the medical application of a kind of antibacterial, relate in particular to a kind of purposes of escherichia coli in the preparation anti-tumor medicine of expressing trail protein matter.

Background technology

Tumor is the principal disease of serious threat human health, has two hang-ups at present on oncotherapy: the one, and effective means of oncotherapy and method.Traditional oncotherapy pattern such as excision, put, chemotherapy etc., it is poor to have therapeutic effect, shortcomings such as side reaction is big, along with the development of molecular biology and genetic engineering, short apoptosis factor many new and effectively Therapeutic Method such as monoclonal antibodies appearred,, angiogenesis inhibitor, the application of cytokine and suicide gene etc.The appearance of these methods, bring new opportunity to oncotherapy, simultaneously, also brought new problem, be the second largest difficult problem on the oncotherapy---the targeting problem of oncotherapy, how to allow new oncotherapy reagent can bring a tremendous development to oncotherapy undoubtedly in the specific more powerful effect of position performance of tumor.Along with deep understanding to antibacterial, it is found that, a lot of antibacterials such as clostruidium, bifidus bacillus, vibrio cholera, Li Site bacterium, salmonella and escherichia coli etc. have the special tumor tissues that is gathered in, and can be in tumor tissues the characteristic of growth and breeding, some antibacterial also has effect (Pawelek J.M, LowK.B, the Bermudes D.Bacteria as tumor-targeting vectors.Lancet Oncol that suppresses tumor growth, 2003,4:548-556.; Zhao Ming, Yang Meng, Xiao Ming, et aI.Tumor-targeting bacterial therapywith amino acid auxotrophs of GFP-expressing Salmonella typhimurium.PNAS, 2005,102:755-760).Utilize this characteristic, started a major transformation of in neoplasm targeted therapy, using about antibacterial.

The Salmonella that the bacillus of anaerobism Pseudomonas, bifidus bacillus and amphimicrobian belong to is used for oncotherapy respectively the deficiency of itself, or is exactly that toxicity is too big, or is exactly the restriction that its tumor-targeting is subjected to the tumor size.So it is the nontoxic restriction that is not subjected to the tumor size again that present exigence is sought a class, the antibacterial that has good tumor-targeting simultaneously again is used for the treatment of tumor aspect.

Escherichia coli (E.coli) belong to gram negative bacilli, are to parasitize the intravital normal flora of human and animal.Colibacillus engineering commonly used at present mainly contains DH5 α, BL21, JM101, JM103～JM109, K802, K803 etc., their genome is stable, genetic background is clear, the more important thing is that it is nontoxic, be widely used in engineered various aspects, as cloning vehicle or some effector gene expression carrier etc.Escherichia coli are applied to the diagnosis and the treatment of tumor, and except that the advantage with antibacterial itself, also have its exclusive characteristics: at first, the Colibacter extracellular bacteria is removed by antibiotic than bacterium in the born of the same parents is easier; Secondly, the non-pathogenic escherichia coli are nontoxic antibacterials, must not carry out attenuation to antibacterial earlier or weak malicious transformation can be used; Once more, more comprehensive to colibacillary genome understanding, it is easier to transform; At last, escherichia coli are facultative anaerobes, can assemble in the necrotic zone of tumor, also can assemble at the abundant position of oxygen, are not subjected in tumor size and the tumor whether anoxybiotic restriction aspect the selection of tumor.

Escherichia coli are also as other antibacterial, have tumor-targeting, when the intravenous injection antibacterial is in the tumor-bearing mice body, annotating second day of bacterium, can in tumor tissues, find germy existence, then not appear in the newspapers but whether escherichia coli itself have the effect that suppresses tumor growth.Research is also found, the mice of in-situ inoculating MCF-7 tumor is after vein gives escherichia coli, escherichia coli can not only arrive the tumor in situ place, also find (Yong A Yu of germy the existence at the metastatic tumor place of offside, Shahrokh Shabahang, Tatyana M Timiryasova, et al.Visualization of tumors and metastases in live animalswith bacteria and vaccinia virus encoding light-emitting proteins.nature biotechnology, 2004,22 (3): 313-321), the prompting escherichia coli the diagnosis of tumor with find to have incomparable advantage.

TRAIL (TNF-related apoptosis inducing ligand) is a member in tumor necrosis factor (TNF) family that finds recently, it is structurally closely similar with FasL, the TRAIL of people and Mus has 281 and 291 aminoacid respectively, and homology reaches 65%.People TRAIL molecular weight is 32.5kD, is II type transmembrane protein, by cytoplasmic domain (14 aminoacid), strides film district (26 aminoacid) and after birth outskirt (241 aminoacid) and forms.Soluble TRAIL molecule (sTrail) is made up of 95～281 amino acids of film outskirt, participates in specific receptors bind, and researcher finds, makes up the protein that clonal expression goes out since 95,104 or 114 residues and all has biologic activity.

TRAIL has stronger short apoptosis effect, experiment in vitro shows, no matter be the film conjunction type or the TRAIL of solubility, the cell generation apoptosis of the special receptor of abduction delivering TRAIL rapidly, its cell death inducing be by with the receptors bind of cell surface, (death domain DD) starts apoptotic pathways by death domain in the born of the same parents of receptor.Up to now, people have found 5 species specificity receptor: TRAIL-R1 (DR4), TRAIL-R2 (DR5), TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and the osteoprotegerin (OPG) of TRAIL.Wherein, DR4 and DR5 are 2 kinds of receptors with complete cytoplasmic region, combine with TRAIL and can cause apoptosis, and DcR1 and DcR2 lack death domain in the complete born of the same parents, be 2 kinds of deficiency membrane receptors, with not transmitting apoptotic signal after the TRAIL combination, and can compete function (the Sheridan JP that suppresses the TRAIL cell death inducing, Marsters SA, Pitti RM.Control of TRAIL-induced apoptosis by a family of signaling and decoyreceptors.Science, 1997,277:818-821.).

Experiment in vitro confirms that TRAIL can kill and wound kinds of tumor cells, as leukaemia, breast cancer cell, colorectal cancer cell, melanoma cell, glioblastoma cell and HIV patient's (Griffith T S such as lymphocyte, Chin W A, Jackson G C.Intracellular regulation of TRAIL-inducedapoptosis in human melanoma cell.J Immunol, 1998,161 (6): 2833～2840).Therefore, TRAIL is considered to a kind of up-and-coming broad-spectrum anti-cancer drug.

TRAIL not only can induce the kinds of tumor cells apoptosis external, confirms also in the experiment that before mice and non-human Primates class animal clinical TRAIL can specific inducing apoptosis of tumour cell, and normal structure and organ are had no side effect.But M.Jo etc. discover that TRAIL can cause the human hepatocytes apoptosis external, and nontoxic to the hepatocyte of rat, mice or Rhesus Macacus, and whether TRAIL can cause that in human body the damage of hepatic injury or other internal organs then still do not have final conclusion.So make TRAIL have even more important biological significance in the effect of the specific short apoptosis of tumor cells of position performance.

At present, escherichia coli and TRAIL are connected, utilize colibacillary tumor-targeting that TRAIL is taken to tumor locus, the antitumor action of bringing into play TRAIL at specific part does not still have report.

Summary of the invention

In order to make TRAIL bring into play its antitumor action at the specific position of tumor, solve the toxic action problem of TRAIL to normal structure and organ, the invention discloses a kind of escherichia coli with tumor-targeting, these escherichia coli are carried trail dna.

The invention also discloses the purposes of escherichia coli in the preparation anti-tumor medicine of above-mentioned expression trail protein matter.

Escherichia coli of the present invention comprise each kind of escherichia coli and genetically engineered colibacillus engineering such as DH5 α, BL21, the JM109 etc. of process thereof.

Escherichia coli of the present invention comprise the carrier that carries trail dna.Trail dna wherein comprises each fragment of TRAIL total length and soluble TRAIL (sTrail), comprises 95～281aa fragment and 114～281aa fragment etc.The TRAIL full length amino acid sequence is shown in sequence in the sequence table 8.The segmental aminoacid sequence of 95～281aa fragment and 114～281aa is shown in sequence in the sequence table 6 and sequence 7.The carrier that wherein carries trail dna is various prokaryotic expression carriers, as pGEX-KG, pET-28a (+) and pBV220 etc.

Tumor of the present invention is various solid tumors, comprises Mus melanin tumor B16-F10-luc-G5, human breast carcinoma MCF-7-luc-F5, people's hepatocarcinoma BEL-7405-luc and SMMC-7721-luc, human prostata cancer PC-3, Mus glioma C6, MBT MB-49 and human fibrosarcoma HT1080 etc.

Colibacillary preparation method of carrying trail dna of the present invention comprises the steps:

1, synthetic primer by the PCR reaction, prepares aforesaid TRAIL total length and sTrail fragment;

2, the TRAIL total length and the sTrail fragment cloning that obtain are gone into above-mentioned prokaryotic expression carrier, obtain recombinant expression carrier;

3, the recombinant expression carrier that obtains is transformed above-mentioned escherichia coli, obtain to carry the trail dna escherichia coli.

Wherein the recombinant prokaryotic expression vector of Huo Deing comprises (95～281aa) carriers or 6His-sTrail fusion rotein pET-sTrail (95～281aa) carriers or by non-fusion rotein pBV220-sTrail (114～281aa) carriers of temperature control abduction delivering by the GST-sTrail fusion rotein pGEX-sTrail of IPTG abduction delivering.

The purposes of escherichia coli in the preparation anti-tumor medicine of trail dna of carrying disclosed by the invention is by following experimental verification.

1, at first prepare mouse tumor model and metastatic tumor model, above-mentioned model is by first culture of tumor cell, then the tumor cell inoculation mice is prepared.Wherein mice is BALB/C mice and nu/nu nude mice, and tumor is above-mentioned various solid tumors;

2, make up photobacteria and detecting then, wherein photobacteria is to make up by luminous plasmid pXen-1 (luxABCDE) is transformed in the escherichia coli, detects by the IVIS imaging system then;

3, observe antibacterial distribution characteristics and tumor-localizing characteristic in animal body.With the photobacteria injection tumor-bearing mice of above-mentioned preparation, observe antibacterial and be distributed in tumor tissues, antibacterial can be located in tumor.

4, with above-mentioned preparation carry trail dna escherichia coli tail vein injection in tumor-bearing mice, observe the effect of escherichia coli to tumor growth.Experimental results show that above-mentioned escherichia coli can delay or suppress tumor growth or tumor is disappeared.

5, the trail dna escherichia coli of carrying of above-mentioned preparation are injected mice metastatic tumor model, prove that it has the ability that is positioned metastatic tumor.

Of the present inventionly carry the trail dna escherichia coli, can specific localization in the tumor tissues of body tumor and transfer, take TRAIL to tumor specific position, the performance antitumor action.Escherichia coli of the present invention add acceptable accessories, to keep this colibacillary activity and stability, can prepare the medicine that is used for diagnosing tumor or treatment.

This medicine can be made the various dosage forms that are suitable for escherichia coli performance antitumor action of the present invention, as tablet, powder, capsule, suppository, injection, suspensoid etc., optimizing injection.Route of administration is that oral, intramuscular injection, intravenous injection etc. are multiple, preferred intravenous injection.

The present invention utilizes colibacillary tumor-targeting, TRAIL is taken to tumor locus, solved the toxic action of TRAIL like this to normal structure and organ, also make TRAIL bring into play stronger, more persistent effect at tumor by local, brought into play colibacillary antitumor action simultaneously, be used to prepare the medicine of diagnosing tumor or treatment, have boundless market prospect.

Description of drawings

Fig. 1 is the scattergram of photobacteria in normal mouse.Wherein A figure is tail vein injection E.coliDH5 α (1 * 10 ⁹The cfu/100ul/ Mus) in the imaging situation of normal mouse after 10～20 minutes, visible photobacteria is distributed in two lungs of mice, and B figure is tail vein injection E.coli DH5 α (1 * 10 ⁹The cfu/100ul/ Mus) the imaging situation after 3 days, visible photobacteria is distributed in the liver of mice, and the distribution situation of other colibacillus engineerings in normal mouse or nude mice is similar therewith.

Fig. 2 is the scattergram of photobacteria in tumor-bearing mice.Wherein A figure is tail vein injection E.coliDH5 α (1 * 10 ⁸The cfu/100ul/ Mus) in lotus the imaging situation of melanoma mice in the time of 1 day arranged, B figure is tail vein injection E.coli DH5 α (1 * 10 ⁸The cfu/100ul/ Mus) in lotus the imaging situation of melanoma mice in the time of 2 days arranged, shown in the circle is the position of mouse tumor inoculation, be that tumor inoculation is subcutaneous in the mice right rear leg, luminous zone is that photobacteria is gathered in tumor tissues, and other colibacillus engineerings have the distribution situation in other solid tumor mices similar therewith melanoma mice or lotus.

Fig. 3 is tail vein injection E.coli DH5 α (1 * 10 ⁸The cfu/100ul/ Mus) anatomy of melanoma mice mice after 13 days is arranged in lotus.Wherein A figure is the exposure status of Different Organs: the 1-liver, and the 2-lungs, the 3-heart, the 4-kidney, the 5-spleen, 6-stomach and intestinal, time of exposure: 300s, all organs are not seen the existence that photobacteria is arranged.B figure is the exposure status of whole tumor, and as seen time of exposure: 30s has the existence of a large amount of photobacterias.C figure is the tumor exposure status of cutting in half, time of exposure: 30s, and from C figure as can be seen: photobacteria mainly is present in the marginal area of tumor, then exists seldom in the intermediary necrotic zone of tumor.Other colibacillus engineerings have the situation in melanoma or other solid tumor mices similar therewith lotus.

Fig. 4 is the inhibitory action figure that bacillus coli DH 5 alpha (GST-sTrail) has the melanoma mouse tumor to grow to lotus, and this engineering bacteria has the effect of other solid tumor mouse tumors similar therewith to lotus.

Fig. 5 is the inhibitory action figure that e. coli bl21 (6his-sTrail) has the melanoma mouse tumor to grow to lotus, and this engineering bacteria has the effect of other solid tumor mouse tumors similar therewith to lotus.

Fig. 6 is the inhibitory action figure that e. coli jm109 (GST-sTrail) has the melanoma mouse tumor to grow to lotus, and this engineering bacteria has the effect of other solid tumor mouse tumors similar therewith to lotus.

Fig. 7 expresses the E.coliDH5 α (1 * 10 that GST-sTrail is arranged for injection ⁸The cfu/100ul/ Mus) situation of melanoma mice tumor growth after 28 days is arranged in lotus.Wherein A figure is the mice that empty carrier group mice is promptly injected E.coliDH5 α (pGEX-KG), long very big of mouse tumor shown in the circle, and occur festering, downright bad phenomenon; B figure is that experimental mice is promptly injected E.coliDH5 α (GST-sTrail)) mice, mouse tumor obviously dwindles shown in the circle, only can see one little melanotic tumor cell subcutaneous.

Fig. 8 is positioned nude mice melanoma lung metastasis for E.coli DH5 α.Wherein A figure takes from the lungs of annotating the melanoma metastasis model nude mice of bacterium after 4 days, has been covered with big and small melanotic tumor metastasis on the visible lung; B figure is the lungs with left figure, and behind the exposure 120s, visible photobacteria is positioned the neoplasm metastasis kitchen range of lungs, and is not having other internal organs of neoplasm metastasis kitchen range, does not then see the existence that photobacteria is arranged.The location situation of other colibacillus engineerings in metastatic tumor is similar therewith.

The specific embodiment

The foundation of embodiment one animal tumor model and metastatic tumor model

One, material:

(1) cell, antibacterial and culture medium

Tumor cell B16-F10-luc-G5, MCF-7-luc-F5, BEL7405-luc and SMMC7721-luc purchase the company in Xenogen, and PC-3, C6, MB-49 and HT1080 purchase the company in ATCC, and E.coli DH5 α, BL21, JM109 are commercial, and this laboratory is preserved.Cell culture medium: RPMI-1640, DMEM, hyclone are Sigma company product.Bacteria culture media: each composition peptone, yeast extract are purchased the company in OXOID among the LB, and sodium chloride is homemade analytical pure.

(2) plasmid, carrier and library

PXen-1 (luxABCDE) plasmid is purchased the company in Xenogen, and pGEX-KG, pET-28a (+) and pBV220 are commercial, is preserved by this laboratory, and people's mammary gland cDNA purchases the company in Clotech in the library.

(3) laboratory animal

BALB/C mice and nu/nu nude mice are provided by Military Medical Science Institute's animal center.BALB/C mice is male, and in ages in nu/nu BALB/C nude mice male and female half and half, 5～6 week, body weight is at 20 ± 5g.

(4) instrument and equipment

IVIS imaging system (Xenogen company); UV-1601 type ultraviolet spectrophotometer (Bio Rad company); Steri-cycle CO ₂Incubator (Thermo ELECTRON company); Low temperature supercentrifuge (Sigma company); The OLYMPUS microscope; PCR instrument (Biometra company)

(5) design of primers

Synthesize following primer by biological engineering Institute for Medical Research of Military Medical Science Institute:

The pGEX-TRAIL design of primers is as follows:

P1: shown in sequence in the sequence table 1; P2: shown in sequence in the sequence table 2.PGEX-sTrail (95～281aa) and pET-sTrail (95～281aa) design of primers are as follows:

P1: shown in sequence in the sequence table 3; P2: with the P2 design of primers of TRAIL total length.Restriction enzyme site is: BamH1 and XholI.

PBV220-sTrail (114～281aa) design of primers is as follows:

P1: shown in sequence in the sequence table 4; P2: shown in sequence in the sequence table 5.

Restriction enzyme site is: EcoRI and PstI.

Two, method

(1) cultivation of tumor cell and preparation

The culture medium constituent and the ratio of B16-F10, MCF-7, PC-3, MB-49 and HT1080 cell are: DMEM 90%, and FBS 10%.The culture medium constituent and the ratio of C6 glioma, BEL7405 and SMMC7721 cell are: RPMI-1640 90%, and FBS 10%.Cell is placed 37 ℃ of 5%CO ₂Cultivate in the incubator, change liquid or go down to posterity according to cell growth state.

Conventional method digestion, collecting cell, the centrifugal 10min of 1200rpm abandons supernatant, and washes cell three times with normal saline, at last with the blood counting chamber counting cells.Cell is resuspended in the normal saline the most at last, and making its final concentration is 5 * 10 ⁷Cell/ml or 5 * 10 ⁶Cell/ml.

(2) foundation of tumor model and metastatic tumor model

BALB/C mice and nude mice tumor model all adopt right rear leg subcutaneous vaccination tumor cell (5 * 10 ⁶Cell/100 μ l/ Mus), the metastasis models of nude mice all adopts the tail vein to inject the mode (5 * 10 of tumor cell ⁵Cell/100 μ l/ Mus).

Three, result:

The tumor model of BALB/C mice and nude mice can form palp tumor behind 7～10 days of inoculated tumour cell; The metastatic tumor model of nude mice forms inject 7～14 days of cell of tumor at vein after, and the metastasis site lung shifts and accounts for more than 80%, and other also can have metastasis as liver, lymph node, fat, bone etc.

Structure and the detection of embodiment two photobacterias

One, material

With embodiment one.

Two, method

(1) preparation of photobacteria

Bacteria culture media LB prepares routinely, bacillus coli DH 5 alpha, and BL21, competent cells such as JM109 prepare routinely.

According to a conventional method pXen-1 (luxABCDE) is transformed into bacillus coli DH 5 alpha respectively, BL21 among the JM109, adopts the IVIS imaging system to observe clone's luminous situation and efficient behind the coated plate.

(2) processing of antibacterial before the experiment

1. in the previous day of annotating bacterium to mice, monoclonal bacterium colony overnight incubation in the LB that has the Amp resistance that the picking luminous efficiency is higher, next day is by 1: 100 this bacterium of switching, continue to shake to the logarithmic growth middle and late stage be OD600=0.6～1.0.

2. press 1OD=8 * 10 ⁸Cfu/ml calculates the bacterium amount, and is centrifugal then, removes LB, and thoroughly gives a baby a bath on the third day after its birth time with physiological saline solution, finally is diluted to 1 * 10 ⁹Cfu/ml.

3. from the bacterium liquid for preparing, get the tail vein injection that 100 μ l are used for mice.

(3) annotate bacterium in tumor-bearing mice

When mouse inoculation tumor after 7～10 days, annotate bacterium in the mode of tail vein injection to mice, annotating the microbial inoculum amount is 1 * 10 ⁸Cfu/100 μ l/ Mus.

Three, result:

By the photobacteria of above method preparation, luminous efficiency is higher, be injected in the animal body after, can under the live body situation, observe antibacterial distribution in animal body, thereby follow the trail of the location (seeing accompanying drawing 1～2) of antibacterial.

Embodiment three antibacterials distribution characteristics and tumor-localizing characteristic research in animal body

One, material:

With embodiment one.

Two, method and result

(1) characteristics that in normal mouse, distribute of antibacterial

Vein gives 2 * 10 ⁹Cfu/100 μ l/ Mus or 1 * 10 ⁹Cfu/100 μ l/ Mus antibacterial is in the intravital 10～20min of normal mouse, and visible antibacterial is distributed in lung, and antibacterial gradually assembles to liver then, and is positioned liver (seeing accompanying drawing 1).Most of mice can not tolerate greater than 1 * 10 ⁹The antibacterial of cfu/100 μ l/ Mus dosage, most mices are in the death in second day of annotating bacterium, and the postmortem result shows: antibacterial mainly is gathered in lungs and the liver of mice, and dead mice postmortem result then shows after dead or a couple of days: antibacterial only is distributed in liver.Intravenous injection 1 * 10 ⁸The mice of cfu/100 μ l/ Mus antibacterial then can all survive, and bacterial distribution finally also only is distributed in liver.

(2) characteristics that in tumor-bearing mice, distribute of antibacterial

Intravenous injection 1 * 10 ⁸Cfu/100 μ l/ Mus antibacterial is in tumor-bearing mice, annotate at the beginning of the bacterium, antibacterial at the intravital distribution of tumor-bearing mice and distributional class in normal mouse seemingly, but at second day that annotates bacterium, the tumor locus of tumor-bearing mice promptly had exist (the seeing accompanying drawing 2) of light.Dissect mice, get normal structure and tumor tissues respectively, find to have only tumor locus that stronger light is arranged, normal structure such as liver, lung, the heart, kidney and gastrointestinal etc. are then unglazed, and prompting: antibacterial all is distributed in tumor tissues.And we find that also for bigger tumor, antibacterial mainly is distributed in the periphery of tumor tissues, the necrotic zone in tumor center, and it is less that antibacterial then distributes, and illustrates that escherichia coli mainly are gathered in the relatively abundanter tumor region (seeing accompanying drawing 3) of oxygen part.Our research is also found, tail vein injection 1 * 10 ⁷The tumor-bearing mice of cfu/100 μ l/ Mus antibacterial after dissecting in second day that annotates bacterium, and tumor locus just has the existence of light, and is then unglazed in normal structure, points out less antibacterial also can locate in tumor.Above result also points out: transforming has the escherichia coli of pXen-1 not change its tumor-targeting.

The structure and the abduction delivering of embodiment four expression vectors

One, material:

With embodiment one.

Two, method and result

(1) pGEX-TRAIL, pGEX-sTrail (95～281aa) and pET-sTrail (95～281aa) clone structure and abduction deliverings

(1) PCR reaction

With people's mammary gland cDNA library is template, carries out the PCR reaction by following condition:

1. 94 ℃ of pre-degeneration 1min; 2. 94 ℃ of degeneration 1min; 3. 55 ℃ annealing 1min; 4. 72 ℃ are extended 1min; 5. 72 ℃ are extended 10min; 6. 4 ℃ stop

2. 3. 4. circulate 30 times, whole reaction system 50 μ l, added enzyme is the Taq archaeal dna polymerase.

(2) structure of efficient expression vector and engineering bacteria

With PCR product electrophoresis, reclaim required fragment, with BamH1 and XholI double digestion, recovery Trail or sTrail fragment are cloned into BamH1 and the XholI site of prokaryotic expression carrier pGEX-KG and pET-28a (+) respectively behind the electrophoresis, be built into pGEX-sTrail (95～281aa) and pET-sTrail (95～281aa) recombinants, difference transformed competence colibacillus bacillus coli DH 5 alpha and BL21, containing picking list bacterium colony on the LB culture plate of resistance screening labelling, after 30 ℃ of shaking tables are cultivated 8h, row bacterium colony PCR, the bacterial strain of criticizing target gene fragment is positive colony.The bacterium liquid of positive colony is transferred in the LB culture fluid, and 30 ℃ of shaking table overnight incubation are extracted plasmid, with BamH1 and XholI double digestion, further identify the purpose bacterial strain, determine positive colony by dna sequencing at last.

(3) abduction delivering of genes of interest

Get engineering bacteria list colony inoculation in 30 ℃ of shaking table overnight incubation of 5ml LB culture fluid (containing the resistance screening labelling), next day, the ratio with 1: 100 was transferred among the fresh LB, continuation is cultured to logarithmic growth mid-term (OD600 is 0.4～0.6) in 30 ℃ of shaking tables, at final concentration is that the IPTG of 0.1mmol/l induces down, 20 ℃ are shaken bacterium 14～16h, centrifugal collection thalline splits bacterium with the ultrasonic bacterium method of splitting, and parallel SDS-PAGE analyzes expression product and expression-form.

Behind the abduction delivering, (95～281aa) SDS-PAGE show pGEX-sTrail: at about 46KD place a newborn band is arranged, and pET-sTrail (95～281aa) SDS-PAGE demonstration: a newborn band is arranged at about 26KD place, expression all accounts for 60% of bacterial protein, and expression-form is the part solubility expression.

(2) pBV220-sTrail (114～281aa) clones' structure and abduction delivering

(1) PCR reaction

(95～281aa) carriers are template, carry out the PCR reaction by above-mentioned condition with pGEX-sTrail.

(2) structure of recombinant vector

With PCR product electrophoresis, reclaim required fragment, and link to each other with the T carrier, connect 2～4h in 16 ℃.

(3) structure of efficient expression vector and engineering bacteria

With T carrier EcoRI and the PstI double digestion after connecting, reclaim the sTrail fragment cloning behind the electrophoresis and go into EcoRI and the PstI site of prokaryotic expression carrier pBV220, be built into the pBV220-sTrail recombinant, transformed competence colibacillus bacillus coli DH 5 alpha or JM109, containing picking list bacterium colony on the LB culture plate of Amp, after 30 ℃ of shaking tables are cultivated 8h, row bacterium colony PCR, the bacterial strain of criticizing target gene fragment is positive colony.The bacterium liquid of positive colony is transferred in the LB culture fluid, and 30 ℃ of shaking table overnight incubation are extracted plasmid, with EcoRI and PstI double digestion, further identify the purpose bacterial strain, determine positive colony by dna sequencing at last.

(4) abduction delivering of genes of interest

Get engineering bacteria list colony inoculation in 5ml LB culture fluid (containing Amp 100mg/L), 30 ℃ of shaking table overnight incubation, next day, the ratio with 1: 100 was transferred in the culture fluid that contains Amp, continuation is cultured to logarithmic growth mid-term (OD600 is 0.4～0.6) in 30 ℃ of shaking tables, be warming up to 39 ℃ of abduction delivering 4h rapidly, centrifugal collection thalline splits bacterium with the ultrasonic bacterium method of splitting, and parallel SDS-PAGE analyzes expression product and expression-form.

Behind the abduction delivering, SDS-PAGE shows: at about 18.5KD place a newborn band is arranged, expression accounts for 20% of bacterial protein, and expression-form is the part solubility expression.

Embodiment five genetic engineering modified bacterium are to the effect of tumor growth

One, material

With embodiment one.

Two, method and result

The 7th～10 day of inoculated tumour, with 15 mices at random be divided into three groups, be respectively the blank group, empty carrier group and experimental group, 5 every group.Wherein the blank group gives 100ul normal saline/Mus, and the empty carrier group gives 1 * 10 respectively ⁸Cfu/100 μ l/ Mus DH5 α (pGEX-KG), DH5 α (pBV220), BL21 (pET-28a (+)) or JM109 (pGEX-KG), the corresponding empty carrier group of experimental group gives 1 * 10 respectively ⁸Cfu/100 μ l/ Mus DH5 α (pGEX-sTrail), DH5 α (pBV220-sTrail), BL21 (pET-sTrail) or JM109 (pGEX-sTrail)), observe the effect of the E.coli of sTrail of carrying to tumor growth.The index of observation is that the growth of mouse tumor volume changes, and the computing formula of gross tumor volume is: V=L * W ²* 0.5 (wherein L is the major axis of tumor, and W is the minor axis of tumor).Statistical method adopts one factor analysis of variance, carries out comparing in twos of sample average, and what method adopted is the Student-Newman-Keuls check, i.e. q check.

Experimental result shows: the growth of mouse tumor volume changes does not have significant difference (P＞0.05) between empty carrier group and matched group, but between experimental group and empty carrier group or and matched group between difference obvious (P＜0.05) then.The mice of the E.coliDH5 α (pGEX-sTrail) of sTrail is carried in intravenous injection, its tumor growth is slow than empty carrier group and matched group, after annotating bacterium the 21st day, in 5 mices of experimental group, have the gross tumor volume of 3 mices to dwindle, flatten comparatively obvious, by the 27th day, tumor almost completely disappeared, and only saw some melanoma cell mouse inoculation tumor site subcutaneous; Though the gross tumor volume of other 2 mices does not disappear fully, the trend that reduces is also arranged, accompanying drawing 7 is seen in the then sustainable growth of gross tumor volume of matched group and empty carrier group mice, and the mouse tumor that has occur the festering phenomenon of necrosis.Point out external evoked escherichia coli expression GST-sTrail, when antibacterial being got in the mice body in intravenous mode, escherichia coli can be taken sTrail to tumor locus specifically, make the powerful antitumor action of its performance.Other carry the engineering bacteria of sTrail such as DH5 α (pBV220-sTrail), BL21 (pET-sTrail), JM109 (pGEX-sTrail) etc. also in various degree the effect that delays or suppress all kinds of tumor growths of mice (seeing accompanying drawing 4～6).

The application of embodiment six genetic engineering modified bacterium in metastatic tumor is found

One, material

With embodiment one.

Two, method and result

Make up metastatic tumor models such as nude mice melanoma, hepatocarcinoma and breast carcinoma as stated above, after 1～2 week, press 0.1ml/10g lumbar injection substrate, behind the living imaging, can judge whether the metastatic tumor model successfully constructs.

After the model construction success, as stated above, the tail vein is annotated bacterium, observes genetic engineering modified E.coli and whether has the ability that is positioned metastatic tumor.

The result: tail vein injection melanoma, tumor cells such as hepatocarcinoma and breast carcinoma are in nude mouse, and the visible lungs metastatic tumor of living imaging successfully constructs after 10 days, and the tail vein injects 1 * 10 ⁸The escherichia coli of cfu/100 μ l/ Mus were dissected the visible lungs of mice and have been covered with the black metastasis that varies in size after 3～4 days, and imaging shows have photobacteria to exist in the lungs metastasis, illustrates that escherichia coli can be positioned the metastasis of tumor (seeing accompanying drawing 8).

Sequence table

＜110〉Biomedicine Analysis Center of Military Medicine Academy, PLA

＜120〉a kind of purposes of escherichia coli in the preparation anti-tumor medicine of expressing trail protein matter

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<213>

<400>2

ccgctcgagt?tagccaacta?aaaaggccc 29

<210>3

<211>29

<212>DNA

<213>

<400>3

cgggatccac?ctctgaggaa?accatttct 29

<210>4

<211>32

<212>DNA

<213>

<400>4

gcgaattcat?ggtgagagaa?agaggtcctc?ag 32

<210>5

<211>30

<212>DNA

<213>

<400>5

cgctgcagtt?agccaactaa?aaaggccccg 30

<210>6

<211>187

<212>PRT

<213>

<400>6

Thr?Ser?Glu?Glu?Thr?Ile?Ser?Thr?Val?Gln?Glu?Lys?Gln?Gln?Asn?Ile

1 5 10 15

Ser?Pro?Leu?Val?Arg?Glu?Arg?Gly?Pro?Gln?Arg?Val?Ala?Ala?His?Ile

20 25 30

Thr?Gly?Thr?Arg?Gly?Arg?Ser?Asn?Thr?Leu?Ser?Ser?Pro?Asn?Ser?Lys

35 40 45

Asn?Glu?Lys?Ala?Leu?Gly?Arg?Lys?Ile?Asn?Ser?Trp?Glu?Ser?Ser?Arg

50 55 60

Ser?Gly?His?Ser?Phe?Leu?Ser?Asn?Leu?His?Leu?Arg?Asn?Gly?Glu?Leu

65 70 75 80

Val?Ile?His?Glu?Lys?Gly?Phe?Tyr?Tyr?Ile?Tyr?Ser?Gln?Thr?Tyr?Phe

85 90 95

Arg?Phe?Gln?Glu?Glu?Ile?Lys?Glu?Asn?Thr?Lys?Asn?Asp?Lys?Gln?Met

100 105 110

Val?Gln?Tyr?Ile?Tyr?Lys?Tyr?Thr?Ser?Tyr?Pro?Asp?Pro?Ile?Leu?Leu

115 120 125

Met?Lys?Ser?Ala?Arg?Asn?Ser?Cys?Trp?Ser?Lys?Asp?Ala?Glu?Tyr?Gly

130 135 140

Leu?Tyr?Ser?Ile?Tyr?Gln?Gly?Gly?Ile?Phe?Glu?Leu?Lys?Glu?Asn?Asp

145 150 155 160

Arg?Ile?Phe?Val?Ser?Val?Thr?Asn?Glu?His?Leu?Ile?Asp?Met?Asp?His

165 170 175

Glu?Ala?Ser?Phe?Phe?Gly?Ala?Phe?Leu?Val?Gly

180 185

<210>7

<211>168

<212>PRT

<213>

<400>7

Val?Arg?Glu?Arg?Gly?Pro?Gln?Arg?Val?Ala?Ala?His?Ile?Thr?Gly?Thr

1 5 10 15

Arg?Gly?Arg?Ser?Asn?Thr?Leu?Ser?Ser?Pro?Asn?Ser?Lys?Asn?Glu?Lys

20 25 30

Ala?Leu?Gly?Arg?Lys?Ile?Asn?Ser?Trp?Glu?Ser?Ser?Arg?Ser?Gly?His

35 40 45

Ser?Phe?Leu?Ser?Asn?Leu?His?Leu?Arg?Asn?Gly?Glu?Leu?Val?Ile?His

50 55 60

Glu?Lys?Gly?Phe?Tyr?Tyr?Ile?Tyr?Ser?Gln?Thr?Tyr?Phe?Arg?Phe?Gln

65 70 75 80

Glu?Glu?Ile?Lys?Glu?Asn?Thr?Lys?Asn?Asp?Lys?Gln?Met?Val?Gln?Tyr

85 90 95

Ile?Tyr?Lys?Tyr?Thr?Ser?Tyr?Pro?Asp?Pro?Ile?Leu?Leu?Met?Lys?Ser

100 105 110

Ala?Arg?Asn?Ser?Cys?Trp?Ser?Lys?Asp?Ala?Glu?Tyr?Gly?Leu?Tyr?Ser

115 120 125

Ile?Tyr?Gln?Gly?Gly?Ile?Phe?Glu?Leu?Lys?Glu?Asn?Asp?Arg?Ile?Phe

130 135 140

Val?Ser?Val?Thr?Asn?Glu?His?Leu?Ile?Asp?Met?Asp?His?Glu?Ala?Ser

145 150 155 160

Phe?Phe?Gly?Ala?Phe?Leu?Val?Gly

165

<210>8

<211>281

<212>PRT

<213>

<400>8

Met?Ala?Met?Met?Glu?Val?Gln?Gly?Gly?Pro?Ser?Leu?Gly?Gln?Thr?Cys

1 5 10 15

Val?Leu?Ile?Val?Ile?Phe?Thr?Val?Leu?Leu?Gln?Ser?Leu?Cys?Val?Ala

20 25 30

Val?Thr?Tyr?Val?Tyr?Phe?Thr?Asn?Glu?Leu?Lys?Gln?Met?Gln?Asp?Lys

35 40 45

Tyr?Ser?Lys?Ser?Gly?Ile?Ala?Cys?Phe?Leu?Lys?Glu?Asp?Asp?Ser?Tyr

50 55 60

Trp?Asp?Pro?Asn?Asp?Glu?Glu?Ser?Met?Asn?Ser?Pro?Cys?Trp?Gln?Val

65 70 75 80

Lys?Trp?Gln?Leu?Arg?Gln?Leu?Val?Arg?Lys?Met?Ile?Leu?Arg?Thr?Ser

85 90 95

Glu?Glu?Thr?Ile?Ser?Thr?Val?Gln?Glu?Lys?Gln?Gln?Asn?Ile?Ser?Pro

100 105 110

Leu?Val?Arg?Glu?Arg?Gly?Pro?Gln?Arg?Val?Ala?Ala?His?Ile?Thr?Gly

115 120 125

Thr?Arg?Gly?Arg?Ser?Asn?Thr?Leu?Ser?Ser?Pro?Asn?Ser?Lys?Asn?Glu

130 135 140

Lys?Ala?Leu?Gly?Arg?Lys?Ile?Asn?Ser?Trp?Glu?Ser?Ser?Arg?Ser?Gly

145 150 155 160

His?Ser?Phe?Leu?Ser?Asn?Leu?His?Leu?Arg?Asn?Gly?Glu?Leu?Val?Ile

165 170 175

His?Glu?Lys?Gly?Phe?Tyr?Tyr?Ile?Tyr?Ser?Gln?Thr?Tyr?Phe?Arg?Phe

180 185 190

Gln?Glu?Glu?Ile?Lys?Glu?Asn?Thr?Lys?Asn?Asp?Lys?Gln?Met?Val?Gln

195 200 205

Tyr?Ile?Tyr?Lys?Tyr?Thr?Ser?Tyr?Pro?Asp?Pro?Ile?Leu?Leu?Met?Lys

210 215 220

Ser?Ala?Arg?Asn?Ser?Cys?Trp?Ser?Lys?Asp?Ala?Glu?Tyr?Gly?Leu?Tyr

225 230 235 240

Ser?Ile?Tyr?Gln?Gly?Gly?Ile?Phe?Glu?Leu?Lys?Glu?Asn?Asp?Arg?Ile

245 250 255

Phe?Val?Ser?Val?Thr?Asn?Glu?His?Leu?Ile?Asp?Met?Asp?His?Glu?Ala

260 265 270

Ser?Phe?Phe?Gly?Ala?Phe?Leu?Val?Gly

275 280

Claims (10)

1, a kind of purposes of escherichia coli in the preparation anti-tumor medicine of expressing trail protein matter.

2, according to the described purposes of claim 1, wherein escherichia coli are each kind of escherichia coli and pass through genetically engineered colibacillus engineering on this basis.

3, according to claim 1 or 2 described purposes, wherein escherichia coli are DH5 α, BL21 or JM109.

4, according to the described purposes of claim 1, wherein escherichia coli comprise the carrier that carries trail dna.

5, according to the described purposes of claim 4, wherein trail dna is each fragment of TRAIL total length or its soluble TRAIL, and the TRAIL full length amino acid sequence is shown in sequence in the sequence table 8.

6, according to the described purposes of claim 5, wherein trail dna is 95～281aa fragment or the 114～281aa fragment of TRAIL, and its aminoacid sequence is respectively shown in sequence in the sequence table 6 and sequence 7.

7, according to the described purposes of claim 4, wherein carrier is a prokaryotic expression carrier.

8, according to the described purposes of claim 7, wherein carrier is pGEX-KG, pET-28a (+) or pBV220.

9, according to the described purposes of claim 1, wherein tumor is a solid tumor.

10, according to claim 1 or 9 described purposes, wherein tumor is Mus melanin tumor B16-F10-luc-G5, human breast carcinoma MCF-7-luc-F5, people's hepatocarcinoma BEL-7405-luc, people's hepatocarcinoma SMMC-7721-luc, human prostata cancer PC-3, Mus glioma C6, MBT MB-49 or human fibrosarcoma cell HT1080.

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