CN106591207B - For express AIF and be integrated with AIF recombinant single chain antibody bacterial strain and the bacterial strain application - Google Patents

Abstract

The present invention provides the applications of the bacterial strain and the bacterial strain of a kind of recombinant single chain antibody for expressing AIF and being integrated with AIF.The characteristic that the technical solution is specifically bound using single-chain antibody T18 and tumor endothelial cell marker molecule TEM1, construct AIF-T18 albumen composition, to realize apoptosis inducing factor AIF to the target administration of tumour cell, its apoptosis-induced effect to tumour cell is promoted.The present invention is integrated with the recombinant plasmid of AIF-T18 expressing gene using pET302 plasmid as vector construction first, is then that host imports recombinant plasmid wherein with prokaryotic expression system E.coli BL21, has obtained with the recombinant bacterial strain for stablizing ability to express.On this basis, the present invention devises efficient protein expression around the characteristic of the recombinant bacterium, and for suitable denaturation, renaturation, purification condition is had matched with protein expressioning product existing for inclusion bodies, to realize effective preparation of AIF-T18.

Description

For express AIF and be integrated with AIF recombinant single chain antibody bacterial strain and the bacterial strain Using

Technical field

The present invention relates to technical field of molecular biology, and it is thin in host to be related to gene recombination technology and recombinant plasmid into one Protein expression in born of the same parents, and in particular to a kind of bacterial strain and the bacterial strain of the recombinant single chain antibody for expressing AIF and being integrated with AIF Application.

Background technique

Endosialin (TEM1/CD248) is I type transmembrane protein, the 80.9KDa's for being glycosylated modification by one Protein core district's groups are at modifying the glycoprotein of after ripening about in 175KDa.TEM1 is the human tumor marker found in the recent period One of, it plays an important role to the cell growth angiogenesis infiltration and transfer of tumour.

TEM1 outer portion is by five globular domains (the c-type Lectin domain of N-terminal, sushi spline structure domain and three tables Skin growth factor (EGF) sample repeat) and a mucoprotein sample district's groups at, wherein core protein a large amount of salivas acidification with thrombus adjusting Albumen is similar.For adult tissue, the expression and distribution of TEM1 be such that uterus > glomerulus > fallopian tubal blood vessel > heart and lung > its Hetero-organization；In addition, TEM1 expression quantity is relatively seldom in endothelial progenitor cells.However, TEM1 has high expression in following tumour, Such as sarcoma, brain tumor, breast cancer, cutaneum carcinoma, colon cancer, and experiment shows in oophoroma, the infiltration of TEM1 expression and tumour, Prognosis is closely related.Therefore, TEM1 is not expressed or low expression in normal tissue, and in the highly expressed spy of neoplasm vascularity Point, the potentiality predictive of TEM1 as the target molecules of tumor diagnosis and therapy.

Apoptosis inducing factor (apoptosis inducing factor, AIF), is that one kind is present in mitochondria Conservative flavoprotein between outer membrane can induce Caspase independence cell apoptosis.The AIF gene of people is located at Xq25- 26, the length for transcribing the mRNA generated is 2.4kb, encodes alternative splicing body of the protein there are three different length of generation, Their amino acid number is respectively 613,609 and 326.In addition to inducible Apoptosis, AIF has both the activity of oxidoreducing enzyme.

Permeability duct after stimulation of the cell by specific apoptosis induction signal, on mitochondrial membrane (mitochondrial permeability transition pores, MPTP) is opened, and AIF is allowed to be discharged into from mitochondria Endochylema, and enter nucleus in and another mitochondrial protein endonuclease G (Endo G) together, cause nuclear dna It is aggregated and fragments into the segment of 50kb size.

In mediating apoptosis process, there are two the effects of aspect by AIF: first is that can be used as the starting of Apoptosis because Son, second is that can be used as the direct effect factor of Apoptosis.Under normal conditions, AIF is present in mitochondria, Ke Yiqing Except intracellular free radical to prevent Apoptosis；After cell is by apoptotic stimulus.AIF comes out from mitochondria first, so After be transferred in cytoplasm, be finally transferred in nucleus play promote Apoptosis effect.

Single-chain antibody T18 in the present invention using screening laboratory early period is as bionavigation, and T18 is anti-using yeast The screening of body library isolate can in combination with the high-affinity of the mankind and muroid TEM1, specific single-chain antibody, by realize its with The amalgamation and expression of humanization toxin AIF, realizes the final purpose of apoptosis of tumor cells.

And two big methods of oncotherapy now are first is that with the immunotherapy of antibody, second is that with the chemotherapy of chemicals Based on.Antibody mediated immunity therapy targeting is strong, but since its relative molecular weight is big, limits the treatment effect of solid tumor Fruit.The chemicals of small molecule have cancer cell high-intensitive fragmentation effect, but due to that cannot differentiate normal cell, to normal Cell also has certain lethal effect, and side effect is obvious.Therefore, antibody clinically is proposed for the treatment of cancer The novel therapeutic agent of one kind of drug conjugates (Antibody Drug Conjugates, ADC), is based on monoclonal antibody skill The effect of specific recognition made of art research combines the high-intensitive lethal effect of chemicals, realizes the good of specific killing tumour Good effect.

Summary of the invention

The present invention is directed to be directed to the technological deficiency of the prior art, a kind of recombination for expressing AIF and being integrated with AIF is provided The application of the bacterial strain of single-chain antibody and the bacterial strain, to solve AIF pairs of apoptosis inducing factor of natural structure in the prior art The attack of tumour cell lacks targeting.

Another technical problem to be solved by the present invention is that lacking in the prior art a kind of for the purpose of assigning AIF targeting AIF- single-chain antibody structural integrity method.

The invention solves another technical problem be to obtain the expression base for being integrated with AIF- single-chain antibody compound It is cumbersome to the expression of such plasmid in the prior art, protein expression level is lower in the case where the recombinant plasmid of cause.

The invention solves another technical problem be for AIF prepared by above method and to be integrated with the weight of AIF Group single-chain antibody, lack in the prior art it is a kind of verifying its to tumor cell specific binding ability and to tumor cytotoxicity imitate The technological means of fruit.

To realize the above technical purpose, the invention adopts the following technical scheme:

A kind of bacterial strain of the recombinant single chain antibody for expressing AIF and being integrated with AIF, which prepared by following methods :

1) take sequence Aif expressing gene as shown in SEQ ID No.1, sequence as shown in SEQ ID No.2 respectively Aif-T18 expressing gene is connect by digestion mode with pET302 plasmid respectively, respectively obtain recombinant plasmid pET302-Aif, pET302-Aif-T18；

2) step 1) resulting recombinant plasmid pET302-Aif, pET302-Aif-T18 are taken respectively, are transferred to E.coli respectively In BL21, respectively obtain recombinant bacterium pET302-Aif-B21 and pET302-Aif-T18-B21, it is as described for express AIF and It is integrated with the bacterial strain of the recombinant single chain antibody of AIF.

Meanwhile the method for preparing AIF the present invention provides application above-mentioned bacterial strains and being integrated with the recombinant single chain antibody of AIF, The following steps are included:

A bacterial strain described in claim 1) is taken to access in LB culture medium with ampicillin, 35~39 DEG C of shake culture mistakes Night；

B step A) is taken) obtained bacterium solution is cultivated, it is seeded to the inoculum concentration of 3~7% (v/v) in fresh LB culture medium, Culture to bacterium solution OD value is 0.6~0.8, and inducer IPTG to 0.8~1.2mM of final concentration is added, and is then shaken in 35~39 DEG C Cultivate 3~5h；

C it) is separated by solid-liquid separation and takes precipitating, thallus is resuspended in the Binding Buffer that 20~25mL is added, and ultrasonication is then solid Liquid separation takes precipitating；

D inclusion body) is collected from the step C) sediment, the Binding Buffer resuspension that 20~25mL is added is forgiven Body；

E step D) is taken) resulting solubilization of inclusion bodies is in albuminous degeneration liquid, until wherein final concentration of the 30 of inclusion body protein ~50mg/ml, room temperature shake 3~5h, are then centrifuged 25~35min with the revolving speed of 12000~14000rpm, supernatant are taken, by it It is added with the volume ratio of 1:80~1:120 into protein renaturation liquid, under agitation, keeps 36~48h in 2~6 DEG C of environment, It mixes with His beads by the volume ratio of 1:3~1:5 after concentration, is then washed using the imidazole solution that concentration is 450~550mM De- albumen recycles PBS solution dialysis.

Preferably, step A) and step C) described in shake culture be revolving speed shaking table culture with 150~250rpm.

Preferably, step B) in for the IPTG that is added into bacterium solution be IPTG that concentration is 1M, by 1/1000 ratio Example is added into the bacterium solution.

Preferably, step C) described in ultrasonication condition are as follows: every ultrasonic treatment 5s stops 8s, so repeat 95 times, Power is 200W, 2 circulations, it is further preferred that the stop condition of ultrasonication are as follows: bacterium solution becomes less viscous thick, more limpid When stop it is broken.

Preferably, step C) in second condition being separated by solid-liquid separation are as follows: the revolving speed centrifugation 8 of 8000~12000rpm~ 12min.It is further preferred that step C) in second be separated by solid-liquid separation after resulting supernatant collection it is whole to 50mL centrifuge tube, in 4 DEG C save.

Preferably, step D) described in collect inclusion body, be first to be washed with Triton X-100/EDTA Solution Precipitating, it is inclusion body that pale precipitation is taken after centrifugation.

Preferably, step D) in Binding Buffer dosage and the dosage phase of Binding Buffer in step C) Deng.

Preferably, step E) described in albuminous degeneration liquid be concentration be 7.5~8.5M urea liquid；The albumen is multiple Property liquid be concentration be 1.8~2.2M urea liquid.

Preferably, step E) described in stirring condition utilize magnetic rotor realize.

Preferably, step E) described in mix be to be stirred 3~5h under the conditions of 2~6 DEG C.

Preferably, step E) continue to execute following operation after the completion: TEM1 negative cells and TEM1 positive cell are taken, are adopted The binding ability of the protein product obtained by flow cytomery and TEM1 negative cells, TEM1 positive cell；Using MTT colorimetric Killing ability of the method detection gained protein product to TEM1 negative cells, TEM1 positive cell.

Preferably, the TEM1 positive cell is prepared by the following method:

M) it obtains TEM1 segment: plasmid pCMV6-XL4-TEM1 being taken to obtain TEM1 segment using glue recycling after double digestion；

N) it constructs- 3Zf (-)-TEM1 recombinant plasmid: the TEM1 segment obtained in step m) is connected by digestion The method connect, is connected toIn -3Zf (-) plasmid to get arrive recombinant plasmid-3Zf(-)-TEM1；

O) it constructs- 3Zf (-)-TEM1-Top10 recombinant bacterial strain: step n) is resulting-3Zf (-)-TEM1 recombinant plasmid is transferred in E.coli Top10, obtains recombinant bacterial strain-3Zf(-)-TEM1-Top10；

P) it constructs pIRES-TEM1-EGFP recombinant plasmid: obtaining from the recombinant bacterial strain obtained in step o) containing TEM1 's- 3Zf (-)-TEM1 recombinant plasmid, by digestion connect method, be connected in pIRES-EGFP plasmid to get To recombinant plasmid pIRES-TEM1-EGFP；

Q) it constructs pIRES-TEM1-EGFP-Top10 recombinant bacterial strain: recombinant plasmid pIRES-TEM1-EGFP is transferred to In E.coli Top10, recombinant bacterial strain pIRES-TEM1-EGFP-Top10 is obtained；

R) construct TEM1 positive MS1 cell: extract from the step q) pIRES-TEM1-EGFP recombinant plasmid electricity rotate into TEM1 negative cells MS1 screens to obtain TEM1 positive cell line by G418.

In above technical scheme, the T18 is the single-chain antibody of specific recognition TEM1 a kind of, the title of the antibody (i.e. T18) belongs to from wound vocabulary.The T18-Aif expressing gene refers to the recombinant single chain antibody T18 for being integrated with Aif expressing gene Expressing gene.As a kind of from wound vocabulary, the technical characteristic of T18-Aif expressing gene is by sequence such as SEQ ID No.2 institute The DNA characterization shown；In the case where knowing particular sequence, which can pass through the synthesis side DNA conventional in the art Method obtains.

The present invention provides a kind of bacterial strain of recombinant single chain antibody for expressing AIF and being integrated with AIF and the bacterial strains Using.Characteristic of the technical solution using single-chain antibody T18 and tumor endothelial cell marker molecule TEM1 specific binding, building AIF-T18 albumen composition, to realize that apoptosis inducing factor AIF to the target administration of tumour cell, promotes it to tumour The apoptosis-induced effect of cell.The present invention is integrated with AIF-T18 expressing gene by vector construction of pET302 plasmid first Recombinant plasmid is then that host imports recombinant plasmid wherein with prokaryotic expression system E.coli BL21, has obtained having and stablize The recombinant bacterial strain of ability to express.On this basis, the present invention devises efficient protein expression side around the characteristic of the recombinant bacterium Method, and for suitable denaturation, renaturation, purification condition is had matched with protein expressioning product existing for inclusion bodies, thus real Effective preparation of AIF-T18 is showed.

At the same time, for investigate above prepared protein product and TEM1 positive cell specific binding capacity and its To the fragmentation effect of tumour cell, the present invention is further provided with the compliance test result step for above-mentioned albumen.

The present invention successfully constructs a kind of recombinant plasmid for being integrated with Aif-T18 expressing fusion protein gene, and is with it Basis has obtained one plant and has stablized expression Aif-T18 fusion protein recombination E.coli bacterial strain.The present invention successfully constructs one plant simultaneously Stable TEM1 positive cell line is laid a good foundation for the verifying of above-mentioned albumen effect.Show the fusion protein pair after verified TEM1 positive cell has the lethal effect of specificity, meets the new concept of ADC medicament research and development, helps to realize specific killing The good result of tumour achievees the purpose that treat tumor disease.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of pET302-Aif-T18 of the present invention Yu pET302-Aif recombinant plasmid；

Fig. 2 is Aif-T18 and Aif protein purification and Western result figure in the specific embodiment of the invention；

Fig. 3 be in the specific embodiment of the invention TEM1 in MS1 cell inner expression fluorescence results figure；

Fig. 4 is TEM1 albumen Western result figure in MS1 and MS1-TEM1 in the specific embodiment of the invention；

Fig. 5 is Aif-T18 fusion protein in the specific embodiment of the invention, the binding ability detection of Aif albumen and cell Result figure

Fig. 6 is Aif-T18 fusion protein in the specific embodiment of the invention, and the lethality of Aif protein on cells detects knot Fruit figure.

Specific embodiment

Below by specific embodiments of the present invention will be described in detail.In order to avoid excessive unnecessary details, In It will not be described in detail in following embodiment to belonging to well known structure or function.In addition to being defined, institute in following embodiment Technical and scientific term has the identical meanings being commonly understood by with those skilled in the art of the invention.

Test reagent consumptive material used in following embodiment is unless otherwise specified conventional biochemical reagent；The experiment Method is unless otherwise specified conventional method；Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result It is averaged；% in following embodiment is unless otherwise instructed mass percentage.

Embodiment 1

The building of 1.1 recombinant expression systems and the expression of Aif, Aif-T18 albumen

(1) chemical synthesis contains the pET302 (SEQ of Aif-T18 (SEQ ID No.2) segment and Aif (SEQ ID No.1) ID No.4) plasmid (as shown in Figure 1), it is transferred in E.coli BL21, picking pET302-Aif-T18 and pET302-Aif Dan Ke Grand access 10mL LB culture medium (containing Amp), 37 DEG C, 200rpm shakes bacterium overnight；

(2) bacterium solution obtained in step (1) is added in new expansion culture medium (200mL LB) in 5% ratio, To bacterium solution OD value between 0.6-0.8, (IPTG concentration is 1M, by 1/1000 addition, bacterium solution for addition inducer IPTG (1/1000) In the final concentration of 1mM of IPTG), 37 DEG C, 200rpm, 4h；

(3) the Binding Buffer of 20-25mL is added, ultrasonication beats 5s, stop 8s, 95 times, power 200W, 2 Circulation.It is become less viscous to bacterium solution thick, stops broken when more limpid, 10000rpm is centrifuged 10min, and supernatant is transferred to newly 50mL centrifuge tube, 4 DEG C preservation.

(4) it is precipitated, the substance of white is washed off, after centrifugation with more washings of Triton X-100/EDTA Solution Pale precipitation is inclusion body, and with the isometric Binding Buffer of thallus is resuspended by inclusion body resuspension, 4 DEG C of ice Case saves.

(5) washed inclusion body Aif-T18 and Aif are dissolved into denaturing liquid (urea of 8M), make its final concentration of 30- 50mg/ml, room temperature rotates 4h on gyroscope, the inclusion body 13000rpm centrifugation 30min of dissolution is removed impurity later, then It is slowly added into renaturation solution (urea of 2M) (1:100), is added portionwise, mixed every time, rotor rotation is added, by renaturation Liquid is concentrated into suitable volumes after being placed in 4 DEG C of renaturation 36-48h, 8000rpm centrifugations, by 1；4 ratio and His beads (1mL) is tied It closes, rotation mixes 4h on 4 DEG C of gyroscopes, is eluted albumen from beads with the imidazoles of 500mM, by the egg after elution It is white to be dialysed with PBS, it is final to obtain Aif-T18 and AIF albumen.As shown in Figure 2.

The building of 1.2TEM1 positive cell line

(1) plasmid pCMV6-XL4 withUsing after EcoRI-HF and XbaI double digestion, glue recycling obtains -3Zf (-) TEM1 segment is connected to by sequence TEM1 segment as shown in SEQ ID No.3 using T4 ligase-3Zf(-) In (SEQ ID No.5), is screened and obtained by amicillin resistance- 3Zf (-)-TEM1 recombinant plasmid, will recombinate Plasmid is transferred in E.coli Top10, obtains recombinant bacterial strain；

(2) by acquisition in step (1)- 3Zf (-)-TEM1 recombinant plasmid and pIRES-EGFP are used After EcoRI-HF and PstI-HF double digestion, TEM1 is connected to pIRES-EGFP after purification, using T4 ligase by digestion products In (SEQ ID No.6), is screened by amicillin resistance and obtain pIRES-TEM1-EGFP recombinant plasmid；It, will after verified Recombinant plasmid pIRES-TEM1-EGFP is converted to E.coli top10, obtains recombinant bacterial strain.

(3) the pIRES-TEM1-EGFP recombinant plasmid obtained is mixed with MS1 cell culture fluid, total volume 500 μL；Using 0.2cm electricity revolving cup, condition setting is 200 Ω 25uF of 1.8KV, and electricity turns time 4.7ms, and acquisition contains pIRES- The MS1 cell of TEM1-EGFP recombinant plasmid, i.e. TEM1 positive cell；Final using the liquid medium-selection containing G418 simultaneously To stable TEM1 positive cell line.Such as Fig. 3 and Fig. 4.

The performance verification of albumen prepared by 1.3 Section 1

A kind of site specific recognition TEM1 kills the fusion protein Aif-T18 function verification method of tumour, with building On the basis of TEM1 positive cell line, Aif albumen and Aif-T18 albumen and TEM1 positive cell is respectively adopted and TEM1 is negative thin Born of the same parents MS1 is incubated for 1-3 hours jointly, uses the binding specificity of flow cytomery Aif albumen and Aif-T18 albumen later, The result shows that Aif albumen does not have the ability of specific binding TEM1 positive cell, and Aif-T18 can specifically bind TEM1 Positive cell.Such as Fig. 5.

A kind of site specific recognition TEM1 kills the fusion protein Aif-T18 function verification method of tumour, with building On the basis of TEM1 positive cell line, Aif albumen and Aif-T18 albumen processing TEM1 positive cell is respectively adopted and TEM1 is negative Use MTT colorimetric determination Aif albumen and Aif-T18 albumen to the killing energy of the specificity of TEM1 positive cell after cell MS1 Power, the results showed that, Aif-T18 energy specific killing TEM1 positive cell, targeting ability is significantly stronger than Aif albumen.Such as Fig. 6.

Embodiment 2

A kind of bacterial strain of the recombinant single chain antibody for expressing AIF and being integrated with AIF, which prepared by following methods :

1) take sequence Aif expressing gene as shown in SEQ ID No.1, sequence as shown in SEQ ID No.2 respectively Aif-T18 expressing gene is connect by digestion mode with pET302 plasmid respectively, respectively obtain recombinant plasmid pET302-Aif, pET302-Aif-T18；

2) step 1) resulting recombinant plasmid pET302-Aif, pET302-Aif-T18 are taken respectively, are transferred to E.coli respectively In BL21, respectively obtain recombinant bacterium pET302-Aif-B21 and pET302-Aif-T18-B21, it is as described for express AIF and It is integrated with the bacterial strain of the recombinant single chain antibody of AIF.

A method of AIF is prepared using above-mentioned bacterial strains and is integrated with the recombinant single chain antibody of AIF, comprising the following steps:

A bacterial strain described in claim 1) is taken to access in LB culture medium with ampicillin, 35 DEG C of shake cultures are stayed overnight；

B step A) is taken) obtained bacterium solution is cultivated, it is seeded in fresh LB culture medium, is trained with the inoculum concentration of 3% (v/v) Supporting to bacterium solution OD value is 0.6, inducer IPTG to final concentration 0.8mM is added, then in 35 DEG C of shake culture 3h；

C it) is separated by solid-liquid separation and takes precipitating, thallus is resuspended in the Binding Buffer that 20mL is added, and ultrasonication, then solid-liquid divides It is precipitated from taking；

D inclusion body) is collected from the step C) sediment, inclusion body is resuspended in the Binding Buffer that 20mL is added；

E step D) is taken) resulting solubilization of inclusion bodies is in albuminous degeneration liquid, until wherein inclusion body protein is final concentration of 30mg/ml, room temperature shake 3h, are then centrifuged 25min with the revolving speed of 12000rpm, take supernatant, it is added with the volume ratio of 1:80 Enter into protein renaturation liquid, keep 36h under agitation, in 2 DEG C of environment, presses the volume ratio of 1:3 after concentration with His beads It mixes, then elutes albumen using the imidazole solution that concentration is 450mM, recycle PBS solution dialysis.

On the basis of above technical scheme, meet the following conditions:

Step C) described in ultrasonication condition are as follows: every ultrasonic treatment 5s stops 8s, so repeats 95 times, power is 200W, 2 circulations.

Step C) in second condition being separated by solid-liquid separation are as follows: the revolving speed of 8000rpm is centrifuged 8min.

Step D) described in collect inclusion body, be first to wash precipitating with Triton X-100/EDTA Solution, centrifugation After take the pale precipitation to be inclusion body.

Step D) in Binding Buffer dosage it is equal with the dosage of Binding Buffer in step C).

Step E) described in albuminous degeneration liquid be concentration be 7.5M urea liquid；The protein renaturation liquid is that concentration is The urea liquid of 1.8M.

Step E) described in mix be to be stirred 3h under the conditions of 2 DEG C.

Step E) continue to execute following operation after the completion: TEM1 negative cells and TEM1 positive cell are taken, it is thin using streaming The binding ability of born of the same parents' instrument detection gained protein product and TEM1 negative cells, TEM1 positive cell；Using MTT colorimetric determination institute Protein product is obtained to the killing ability of TEM1 negative cells, TEM1 positive cell.

The TEM1 positive cell is prepared by the following method:

M) it obtains TEM1 segment: plasmid pCMV6-XL4-TEM1 being taken to obtain TEM1 segment using glue recycling after double digestion；

N) it constructs- 3Zf (-)-TEM1 recombinant plasmid: the TEM1 segment obtained in step m) is connected by digestion The method connect, is connected toIn -3Zf (-) plasmid to get arrive recombinant plasmid-3Zf(-)-TEM1；

O) it constructs- 3Zf (-)-TEM1-Top10 recombinant bacterial strain: step n) is resulting-3Zf (-)-TEM1 recombinant plasmid is transferred in E.coli Top10, obtains recombinant bacterial strain-3Zf(-)-TEM1-Top10；

P) it constructs pIRES-TEM1-EGFP recombinant plasmid: obtaining from the recombinant bacterial strain obtained in step o) containing TEM1 's- 3Zf (-)-TEM1 recombinant plasmid, by digestion connect method, be connected in pIRES-EGFP plasmid to get To recombinant plasmid pIRES-TEM1-EGFP；

Q) it constructs pIRES-TEM1-EGFP-Top10 recombinant bacterial strain: recombinant plasmid pIRES-TEM1-EGFP is transferred to In E.coli Top10, recombinant bacterial strain pIRES-TEM1-EGFP-Top10 is obtained；

R) construct TEM1 positive MS1 cell: extract from the step q) pIRES-TEM1-EGFP recombinant plasmid electricity rotate into TEM1 negative cells MS1 screens to obtain TEM1 positive cell line by G418.

Embodiment 3

A kind of bacterial strain of the recombinant single chain antibody for expressing AIF and being integrated with AIF, which prepared by following methods :

1) take sequence Aif expressing gene as shown in SEQ ID No.1, sequence as shown in SEQ ID No.2 respectively Aif-T18 expressing gene is connect by digestion mode with pET302 plasmid respectively, respectively obtain recombinant plasmid pET302-Aif, pET302-Aif-T18；

2) step 1) resulting recombinant plasmid pET302-Aif, pET302-Aif-T18 are taken respectively, are transferred to E.coli respectively In BL21, respectively obtain recombinant bacterium pET302-Aif-B21 and pET302-Aif-T18-B21, it is as described for express AIF and It is integrated with the bacterial strain of the recombinant single chain antibody of AIF.

A method of AIF is prepared using above-mentioned bacterial strains and is integrated with the recombinant single chain antibody of AIF, comprising the following steps:

A bacterial strain described in claim 1) is taken to access in LB culture medium with ampicillin, 39 DEG C of shake cultures are stayed overnight；

B step A) is taken) obtained bacterium solution is cultivated, it is seeded in fresh LB culture medium, is trained with the inoculum concentration of 7% (v/v) Supporting to bacterium solution OD value is 0.8, inducer IPTG to final concentration 1.2mM is added, then in 39 DEG C of shake culture 5h；

C it) is separated by solid-liquid separation and takes precipitating, thallus is resuspended in the Binding Buffer that 25mL is added, and ultrasonication, then solid-liquid divides It is precipitated from taking；

D inclusion body) is collected from the step C) sediment, inclusion body is resuspended in the Binding Buffer that 25mL is added；

E step D) is taken) resulting solubilization of inclusion bodies is in albuminous degeneration liquid, until wherein inclusion body protein is final concentration of 50mg/ml, room temperature shake 5h, are then centrifuged 35min with the revolving speed of 14000rpm, take supernatant, it is added with the volume ratio of 1:120 Enter into protein renaturation liquid, keep 48h under agitation, in 6 DEG C of environment, presses the volume ratio of 1:5 after concentration with His beads It mixes, then elutes albumen using the imidazole solution that concentration is 550mM, recycle PBS solution dialysis.

On the basis of above technical scheme, meet the following conditions:

Step C) in second condition being separated by solid-liquid separation are as follows: the revolving speed of 12000rpm is centrifuged 12min.

Step E) described in albuminous degeneration liquid be concentration be 8.5M urea liquid；The protein renaturation liquid is that concentration is The urea liquid of 2.2M.

Step E) described in mix be to be stirred 5h under the conditions of 6 DEG C.

Step E) continue to execute following operation after the completion: TEM1 negative cells and TEM1 positive cell are taken, it is thin using streaming The binding ability of born of the same parents' instrument detection gained protein product and TEM1 negative cells, TEM1 positive cell；Using MTT colorimetric determination institute Protein product is obtained to the killing ability of TEM1 negative cells, TEM1 positive cell.

Embodiment 4

A kind of bacterial strain of the recombinant single chain antibody for expressing AIF and being integrated with AIF, which prepared by following methods :

1) take sequence Aif expressing gene as shown in SEQ ID No.1, sequence as shown in SEQ ID No.2 respectively Aif-T18 expressing gene is connect by digestion mode with pET302 plasmid respectively, respectively obtain recombinant plasmid pET302-Aif, pET302-Aif-T18；

2) step 1) resulting recombinant plasmid pET302-Aif, pET302-Aif-T18 are taken respectively, are transferred to E.coli respectively In BL21, respectively obtain recombinant bacterium pET302-Aif-B21 and pET302-Aif-T18-B21, it is as described for express AIF and It is integrated with the bacterial strain of the recombinant single chain antibody of AIF.

A method of AIF is prepared using above-mentioned bacterial strains and is integrated with the recombinant single chain antibody of AIF, comprising the following steps:

A bacterial strain described in claim 1) is taken to access in LB culture medium with ampicillin, 37 DEG C of shake cultures are stayed overnight；

B step A) is taken) obtained bacterium solution is cultivated, it is seeded in fresh LB culture medium, is trained with the inoculum concentration of 5% (v/v) Supporting to bacterium solution OD value is 0.7, inducer IPTG to final concentration 1mM is added, then in 37 DEG C of shake culture 4h；

C it) is separated by solid-liquid separation and takes precipitating, thallus is resuspended in the Binding Buffer that 22mL is added, and ultrasonication, then solid-liquid divides It is precipitated from taking；

D inclusion body) is collected from the step C) sediment, inclusion body is resuspended in the Binding Buffer that 22mL is added；

E step D) is taken) resulting solubilization of inclusion bodies is in albuminous degeneration liquid, until wherein inclusion body protein is final concentration of 40mg/ml, room temperature shake 4h, are then centrifuged 30min with the revolving speed of 13000rpm, take supernatant, it is added with the volume ratio of 1:100 Enter into protein renaturation liquid, keep 42h under agitation, in 4 DEG C of environment, presses the volume ratio of 1:4 after concentration with His beads It mixes, then elutes albumen using the imidazole solution that concentration is 500mM, recycle PBS solution dialysis.

The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention, It is not intended to limit the invention.All any modifications, equivalent replacements, and improvements etc. done in application range of the invention, should all It is included within protection scope of the present invention.

SEQUENCE LISTING

<110>University Of Nanchang

<120>for express AIF and be integrated with AIF recombinant single chain antibody bacterial strain and the bacterial strain application

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 1536

<212> DNA

<213>artificial sequence

<400> 1

ttagggctga caccagaaca gaaacagaaa aaggccgcgt tatctgcttc agaaggagag 60

gaagttcctc aagacaaggc gccaagtcat gttcctttcc tgctaattgg tggaggcaca 120

gctgcttttg ctgcagccag atccatccgg gctcgggatc ctggggccag ggtactgatt 180

gtatctgaag atcctgagct gccgtacatg cgacctcctc tttcaaaaga actgtggttt 240

tcagatgacc caaatgtcac aaagacactg cgattcaaac agtggaatgg aaaagagaga 300

agcatatatt tccagccacc ttctttctat gtctctgctc aggacctgcc tcatattgag 360

aatggtggtg tggctgtcct cactgggaag aaggtagtac agctggatgt gagagacaac 420

atggtgaaac ttaatgatgg ctctcaaata acctatgaaa agtgcttgat tgcaacagga 480

ggtactccaa gaagtctgtc tgccattgat agggctggag cagaggtgaa gagtagaaca 540

acgcttttca gaaagattgg agactttaga agcttggaga agatttcacg ggaagtcaaa 600

tcaattacga ttatcggtgg gggcttcctt ggtagcgaac tggcctgtgc tcttggcaga 660

aaggctcgag ccttgggcac agaagtgatt caactcttcc ccgagaaagg aaatatggga 720

aagatcctcc ccgaatacct cagcaactgg accatggaaa aagtcagacg agagggggtt 780

aaggtgatgc ccaatgctat tgtgcaatcc gttggagtca gcagtggcaa gttacttatc 840

aagctgaaag acggcaggaa ggtagaaact gaccacatag tggcagctgt gggcctggag 900

cccaatgttg agttggccaa gactggtggc ctggaaatag actcagattt tggtggcttc 960

cgggtaaatg cagagctaca agcacgctct aacatctggg tggcaggaga tgctgcatgc 1020

ttctacgata taaagttggg aaggaggcgg gtagagcacc atgatcacgc tgttgtgagt 1080

ggaagattgg ctggagaaaa tatgactgga gctgctaagc cgtactggca tcagtcaatg 1140

ttctggagtg atttgggccc cgatgttggc tatgaagcta ttggtcttgt ggacagtagt 1200

ttgcccacag ttggtgtttt tgcaaaagca actgcacaag acaaccccaa atctgccaca 1260

gagcagtcag gaactggtat ccgatcagag agtgagacag agtccgaggc ctcagaaatt 1320

actattcctc ccagcacccc ggcagttcca caggctcccg tccaggggga ggactacggc 1380

aaaggtgtca tcttctacct cagggacaaa gtggtcgtgg ggattgtgct atggaacatc 1440

tttaaccgaa tgccaatagc aaggaagatc attaaggacg gtgagcagca tgaagatctc 1500

aatgaagtag ccaaactatt caacattcat gaagac 1536

<210> 2

<211> 2376

<212> DNA

<213>artificial sequence

<400> 2

ttagggctga caccagaaca gaaacagaaa aaggccgcgt tatctgcttc agaaggagag 60

gaagttcctc aagacaaggc gccaagtcat gttcctttcc tgctaattgg tggaggcaca 120

gctgcttttg ctgcagccag atccatccgg gctcgggatc ctggggccag ggtactgatt 180

gtatctgaag atcctgagct gccgtacatg cgacctcctc tttcaaaaga actgtggttt 240

tcagatgacc caaatgtcac aaagacactg cgattcaaac agtggaatgg aaaagagaga 300

agcatatatt tccagccacc ttctttctat gtctctgctc aggacctgcc tcatattgag 360

aatggtggtg tggctgtcct cactgggaag aaggtagtac agctggatgt gagagacaac 420

atggtgaaac ttaatgatgg ctctcaaata acctatgaaa agtgcttgat tgcaacagga 480

ggtactccaa gaagtctgtc tgccattgat agggctggag cagaggtgaa gagtagaaca 540

acgcttttca gaaagattgg agactttaga agcttggaga agatttcacg ggaagtcaaa 600

tcaattacga ttatcggtgg gggcttcctt ggtagcgaac tggcctgtgc tcttggcaga 660

aaggctcgag ccttgggcac agaagtgatt caactcttcc ccgagaaagg aaatatggga 720

aagatcctcc ccgaatacct cagcaactgg accatggaaa aagtcagacg agagggggtt 780

aaggtgatgc ccaatgctat tgtgcaatcc gttggagtca gcagtggcaa gttacttatc 840

aagctgaaag acggcaggaa ggtagaaact gaccacatag tggcagctgt gggcctggag 900

cccaatgttg agttggccaa gactggtggc ctggaaatag actcagattt tggtggcttc 960

cgggtaaatg cagagctaca agcacgctct aacatctggg tggcaggaga tgctgcatgc 1020

ttctacgata taaagttggg aaggaggcgg gtagagcacc atgatcacgc tgttgtgagt 1080

ggaagattgg ctggagaaaa tatgactgga gctgctaagc cgtactggca tcagtcaatg 1140

ttctggagtg atttgggccc cgatgttggc tatgaagcta ttggtcttgt ggacagtagt 1200

ttgcccacag ttggtgtttt tgcaaaagca actgcacaag acaaccccaa atctgccaca 1260

gagcagtcag gaactggtat ccgatcagag agtgagacag agtccgaggc ctcagaaatt 1320

actattcctc ccagcacccc ggcagttcca caggctcccg tccaggggga ggactacggc 1380

aaaggtgtca tcttctacct cagggacaaa gtggtcgtgg ggattgtgct atggaacatc 1440

tttaaccgaa tgccaatagc aaggaagatc attaaggacg gtgagcagca tgaagatctc 1500

aatgaagtag ccaaactatt caacattcat gaagacgaat tcgcgggtaa tcgtgtgcgt 1560

cgctctgttg gtcttaaggg tggtggaggt ggttctggtg gtggaggttc tggtggtggt 1620

ggatctggcc agcttgtgct gactcagcca ccttccctct ctgcatctcc tggagcatca 1680

gccagtctca cctgcacctt acgcagtgac atcaatgttg gttcctacag gatatcctgg 1740

taccagcaga agccagggag tcctccccag tatctcctga gctacaaatc agactcagat 1800

aagcagaagg gctctggagt ccccagccgc ttctctggat ccaaagatgc ttcggccaat 1860

gcagggattt tactcatctc tgggctccag tctgaggatg aggctgacta ttattgtatg 1920

atttggcaca acagcgctgg ggtgttcggc gggggcacca agctgaccgt cctaggcggt 1980

ggttcctcta gatcttcctc ctctggtggc ggtggctcgg gcggtggtgg gcaggtgcag 2040

ctgcaggagt cgggaggaac cttggtacag cctggggggt ccctgagact ctcttgtgaa 2100

gcctctggat tcacctttag caactatgcc atgggctggg tccgccagac tccaggaaag 2160

gggctggagt ggctgtcggc tattcgtaaa agtggcacta ccacatacta cgcggactcc 2220

gtgaagggcc ggttcatcat ctccagagac aattccaaga acaccctgta tctgcaaatg 2280

aataggctga gagtcggcga cacggccact tattactgtg cgactcaccc catcgcgggc 2340

tactggggcc agggaagcct ggtcactgtc tcctcc 2376

<210> 3

<211> 2576

<212> DNA

<213>artificial sequence

<400> 3

agtccggggg catcgcgatg ctgctgcgcc tgttgctggc ctgggcggcc gcagggccca 60

cactgggcca ggacccctgg gctgctgagc cccgtgccgc ctgcggcccc agcagctgct 120

acgctctctt cccacggcgc cgcaccttcc tggaggcctg gcgggcctgc cgcgagctgg 180

ggggcgacct ggccactcct cggacccccg aggaggccca gcgtgtggac agcctggtgg 240

gtgcgggccc agccagccgg ctgctgtgga tcgggctgca gcggcaggcc cggcaatgcc 300

agctgcagcg cccactgcgc ggcttcacgt ggaccacagg ggaccaggac acggctttca 360

ccaactgggc ccagccagcc tctggaggcc cctgcccggc ccagcgctgt gtggccctgg 420

aggcaagtgg cgagcaccgc tggctggagg gctcgtgcac gctggctgtc gacggctacc 480

tgtgccagtt tggcttcgag ggcgcctgcc cggcgctgca agatgaggcg ggccaggccg 540

gcccagccgt gtataccacg cccttccacc tggtctccac agagtttgag tggctgccct 600

tcggctctgt ggccgctgtg cagtgccagg ctggcagggg agcctctctg ctctgcgtga 660

agcagcctga gggaggtgtg ggctggtcac gggctgggcc cctgtgcctg gggactggct 720

gcagccctga caacgggggc tgcgaacacg aatgtgtgga ggaggtggat ggtcacgtgt 780

cctgccgctg cactgagggc ttccggctgg cagcagacgg gcgcagttgc gaggacccct 840

gtgcccaggc tccgtgcgag cagcagtgtg agcccggtgg gccacaaggc tacagctgcc 900

actgtcgcct gggtttccgg ccagcggagg atgatccgca ccgctgtgtg gacacagatg 960

agtgccagat tgccggtgtg tgccagcaga tgtgtgtcaa ctacgttggt ggcttcgagt 1020

gttattgtag cgagggacat gagctggagg ctgatggcat cagctgcagc cctgcagggg 1080

ccatgggtgc ccaggcttcc caggacctcg gagatgagtt gctggatgac ggggaggatg 1140

aggaagatga agacgaggcc tggaaggcct tcaacggtgg ctggacggag atgcctggga 1200

tcctgtggat ggagcctacg cagccgcctg actttgccct ggcctataga ccgagcttcc 1260

cagaggacag agagccacag ataccctacc cggagcccac ctggccaccc ccgctcagtg 1320

cccccagggt cccctaccac tcctcagtgc tctccgtcac ccggcctgtg gtggtctctg 1380

ccacgcatcc cacactgcct tctgcccacc agcctcctgt gatccctgcc acacacccag 1440

ctttgtcccg tgaccaccag atccccgtga tcgcagccaa ctatccagat ctgccttctg 1500

cctaccaacc cggtattctc tctgtctctc attcagcaca gcctcctgcc caccagcccc 1560

ctatgatctc aaccaaatat ccggagctct tccctgccca ccagtccccc atgtttccag 1620

acacccgggt cgctggcacc cagaccacca ctcatttgcc tggaatccca cctaaccatg 1680

cccctctggt caccaccctc ggtgcccagc taccccctca agccccagat gcccttgtcc 1740

tcagaaccca ggccacccag cttcccatta tcccaactgc ccagccctct ctgaccacca 1800

cctccaggtc ccctgtgtct cctgcccatc aaatctctgt gcctgctgcc acccagcccg 1860

cagccctccc caccctcctg ccctctcaga gccccactaa ccagacctca cccatcagcc 1920

ctacacatcc ccattccaaa gccccccaaa tcccaaggga agatggcccc agtcccaagt 1980

tggccctgtg gctgccctca ccagctccca cagcagcccc aacagccctg ggggaggctg 2040

gtcttgccga gcacagccag agggatgacc ggtggctgct ggtggcactc ctggtgccaa 2100

cgtgtgtctt tttggtggtc ctgcttgcac tgggcatcgt gtactgcacc cgctgtggcc 2160

cccatgcacc caacaagcgc atcactgact gctatcgctg ggtcatccat gctgggagca 2220

agagcccaac agaacccatg ccccccaggg gcagcctcac aggggtgcag acctgcagaa 2280

ccagcgtgtg atggggtgca gacccccctc atggagtatg gggcgctgga cacatggccg 2340

gggctgcacc agggacccat gggggctgcc cagctggaca gatggcttcc tgctccccag 2400

gcccagccag ggtcctctct caaccactag acttggctct caggaactct gcttcctggc 2460

ccagcgctcg tgaccaagga tacaccaaag cccttaagac ctcagggggc gggtgctggg 2520

gtcttctcca ataaatgggg tgtcaacctt acccaaggaa aaaaaaaaaa aaaaaa 2576

<210> 4

<211> 5712

<212> DNA

<213>artificial sequence

<400> 4

gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat 60

tcccctctag aaataatttt gtttaaactt taagaaggag atatacatat gcatcatcat 120

catcatcacg tgaattcgct cgagatcgat gatattcgag cctaggtata atcggatccg 180

gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta 240

gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact 300

atatccggat atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag 360

catccagggt gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg 420

cctgactgcg ttagcaattt aactgtgata aactaccgca ttaaagctag cttatcgatg 480

ataagctgtc aaacatgaga attaattctt gaagacgaaa gggcctcgtg atacgcctat 540

ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg 600

gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc 660

tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta 720

ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg 780

ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg 840

gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac 900

gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtgttg 960

acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt 1020

actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg 1080

ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac 1140

cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt 1200

gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgcag 1260

caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc 1320

aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc 1380

ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta 1440

tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg 1500

ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga 1560

ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac 1620

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 1680

tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 1740

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 1800

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 1860

gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 1920

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 1980

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 2040

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 2100

cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 2160

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 2220

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 2280

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 2340

gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 2400

ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg 2460

ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc 2520

tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct 2580

cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt 2640

gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct 2700

tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt 2760

cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg 2820

tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc 2880

tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc 2940

tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg 3000

atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg 3060

gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact 3120

cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag 3180

catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga 3240

ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg 3300

cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg 3360

caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc 3420

caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg 3480

gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct 3540

ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg 3600

tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc 3660

ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga 3720

tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc 3780

cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc 3840

cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca 3900

gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga 3960

aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata 4020

ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga 4080

cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg 4140

cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca 4200

agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc 4260

gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4320

aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt 4380

gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg 4440

tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata 4500

taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc 4560

agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc 4620

atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg 4680

gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta 4740

tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg 4800

atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg 4860

gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca 4920

ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc 4980

agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg 5040

cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc 5100

gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc 5160

aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc 5220

atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg 5280

cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt 5340

ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag 5400

gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat 5460

taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc 5520

atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc 5580

gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc 5640

gatataggcg ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc 5700

gtagaggatc ga 5712

<210> 5

<211> 3197

<212> DNA

<213>artificial sequence

<400> 5

gggcgaattc gagctcggta cccggggatc ctctagagtc gacctgcagg catgcaagct 60

tgagtattct atagtgtcac ctaaatagct tggcgtaatc atggtcatag ctgtttcctg 120

tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 180

aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 240

ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 300

gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 360

tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 420

aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 480

gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 540

aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 600

ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 660

tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 720

tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 780

ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 840

tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 900

ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 960

tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 1020

aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 1080

aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 1140

aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 1200

ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 1260

acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 1320

ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 1380

gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 1440

taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 1500

tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 1560

gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 1620

cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 1680

aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 1740

cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 1800

tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 1860

gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 1920

tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 1980

gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 2040

ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 2100

cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 2160

agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 2220

gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca 2280

tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg 2340

atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag 2400

cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg 2460

gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg 2520

aaataccgca cagatgcgta aggagaaaat accgcatcag gacgcgccct gtagcggcgc 2580

attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 2640

agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 2700

tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 2760

ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 2820

ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 2880

aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 2940

ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt ttaacaaaat 3000

attaacgctt acaatttcca ttcgccattc aggctgcgca actgttggga agggcgatcg 3060

gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta 3120

agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattg 3180

taatacgact cactata 3197

<210> 6

<211> 5162

<212> DNA

<213>artificial sequence

<400> 6

gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 900

gagctcggat cgatatctgc ggccgcgtcg acggaattca gtggatccac tagtaacggc 960

cgccagtgtg ctggaattaa ttcgctgtct gcgagggcca gctgttgggg tgagtactcc 1020

ctctcaaaag cgggcatgac ttctgcgcta agattgtcag tttccaaaaa cgaggaggat 1080

ttgatattca cctggcccgc ggtgatgcct ttgagggtgg ccgcgtccat ctggtcagaa 1140

aagacaatct ttttgttgtc aagcttgagg tgtggcaggc ttgagatctg gccatacact 1200

tgagtgacaa tgacatccac tttgcctttc tctccacagg tgtccactcc caggtccaac 1260

tgcaggtcga gcatgcatct agggcggcca attccgcccc tctccctccc ccccccctaa 1320

cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgtgattttc 1380

caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac 1440

gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt 1500

gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg 1560

caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata 1620

agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga 1680

aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt 1740

accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc 1800

gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac 1860

acgatgataa gcttgccaca acccgggatc caccggtcgc caccatggtg agcaagggcg 1920

aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 1980

acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 2040

agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 2100

cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 2160

agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 2220

actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 2280

tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 2340

acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 2400

tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 2460

acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 2520

ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 2580

ccgccgccgg gatcactctc ggcatggacg agctgtacaa gtaaagcggc cctagagctc 2640

gctgatcagc ctcgactgtg cctctagttg ccagccatct gttgtttgcc cctcccccgt 2700

gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 2760

tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 2820

caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 2880

ttctgaggcg gaaagaacca gctggggctc gagtgcattc tagttgtggt ttgtccaaac 2940

tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat 3000

catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac 3060

gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 3120

ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat 3180

gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc 3240

tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 3300

cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 3360

gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 3420

gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3480

gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga 3540

ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc 3600

aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3660

tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3720

ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca 3780

gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3840

ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag 3900

ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca 3960

agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 4020

ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 4080

aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta 4140

tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 4200

cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga 4260

tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac 4320

cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc 4380

ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 4440

gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4500

gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4560

gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa 4620

gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg 4680

tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4740

aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4800

cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4860

caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4920

cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg 4980

ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc 5040

aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta 5100

tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 5160

tc 5162

Claims (10)

1. a kind of for expressing the bacterial strain for being integrated with the recombinant single chain antibody of AIF, it is characterised in that the bacterial strain is by following methods Preparation:

1) sequence Aif-T18 expressing gene as shown in SEQ ID No.2 is taken, is connect by digestion mode with pET302 plasmid, Obtain recombinant plasmid pET302-Aif-T18；

2) the resulting recombinant plasmid pET302-Aif-T18 of step 1) is taken, is transferred in E.coli BL21, obtains recombinant bacterium PET302-Aif-T18-B21, it is as described for expressing the bacterial strain for the recombinant single chain antibody for being integrated with AIF.

2. application claim 1 described in bacterial strain preparation be integrated with AIF recombinant single chain antibody method, it is characterised in that including with Lower step:

A bacterial strain described in claim 1) is taken to access in LB culture medium with ampicillin, 35~39 DEG C of shake cultures are stayed overnight；

B step A) is taken) obtained bacterium solution is cultivated, it is seeded in fresh LB culture medium, is cultivated with the inoculum concentration of 3~7% (v/v) It is 0.6~0.8 to bacterium solution OD value, inducer IPTG to 0.8~1.2mM of final concentration is added, then in 35~39 DEG C of shake cultures 3 ~5h；

C it) is separated by solid-liquid separation and takes precipitating, thallus is resuspended in the Binding Buffer that 20~25mL is added, and ultrasonication, then solid-liquid divides It is precipitated from taking；

D inclusion body) is collected from the step C) sediment, inclusion body is resuspended in the Binding Buffer that 20~25mL is added；

E) take step D) resulting solubilization of inclusion bodies in albuminous degeneration liquid, until wherein final concentration of the 30 of inclusion body protein~ 50mg/mL, room temperature shake 3~5h, are then centrifuged 25~35min with the revolving speed of 12000~14000rpm, take supernatant, by its with The volume ratio of 1:80~1:120 is added into protein renaturation liquid, under agitation, 36~48h is kept in 2~6 DEG C of environment, dense It mixes with His beads by the volume ratio of 1:3~1:5 after contracting, is then eluted using the imidazole solution that concentration is 450~550mM Albumen recycles PBS solution dialysis.

3. according to the method described in claim 2, it is characterized in that step C) described in ultrasonication condition are as follows: at every ultrasound 5s is managed, 8s is stopped, is so repeated 95 times, power 200W, 2 circulations.

4. according to the method described in claim 2, it is characterized in that step C) in second of condition being separated by solid-liquid separation are as follows: 8000~ The revolving speed of 12000rpm is centrifuged 8~12min.

5. according to the method described in claim 2, it is characterized in that step D) described in collect inclusion body, be first to use Triton X-100/EDTA Solution washing precipitating, it is inclusion body that pale precipitation is taken after centrifugation.

6. according to the method described in claim 2, it is characterized in that step D) in Binding Buffer dosage and step C) The dosage of middle Binding Buffer is equal.

7. according to the method described in claim 2, it is characterized in that step E) described in albuminous degeneration liquid be concentration be 7.5~ The urea liquid of 8.5M；The protein renaturation liquid is the urea liquid that concentration is 1.8~2.2M.

8. according to the method described in claim 2, it is characterized in that step E) described in mix stirred under the conditions of 2~6 DEG C Mix 3~5h.

9. according to the method described in claim 2, it is characterized in that step E) continue to execute following operation after the completion: take TEM1 negative Property cell and TEM1 positive cell, it is positive thin using protein product obtained by flow cytomery and TEM1 negative cells, TEM1 The binding ability of born of the same parents；Using protein product obtained by MTT colorimetric determination to the killing energy of TEM1 negative cells, TEM1 positive cell Power.

10. according to the method described in claim 9, it is characterized in that the TEM1 positive cell is to be prepared by the following method :

M) it obtains TEM1 segment: plasmid pCMV6-XL4-TEM1 being taken to obtain TEM1 segment using glue recycling after double digestion；

N) it constructs- 3Zf (-)-TEM1 recombinant plasmid: the TEM1 segment obtained in step m) is connected by digestion Method is connected toIn -3Zf (-) plasmid to get arrive recombinant plasmid-3Zf(-)-TEM1；

O) it constructs- 3Zf (-)-TEM1-Top10 recombinant bacterial strain: step n) is resulting-3Zf(-)-TEM1 Recombinant plasmid is transferred in E.coli Top10, obtains recombinant bacterial strain-3Zf(-)-TEM1-Top10；

P) it constructs pIRES-TEM1-EGFP recombinant plasmid: obtaining from the recombinant bacterial strain obtained in step o) containing TEM1's- 3Zf (-)-TEM1 recombinant plasmid, the method connected by digestion, is connected in pIRES-EGFP plasmid to get arriving Recombinant plasmid pIRES-TEM1-EGFP；

Q) it constructs pIRES-TEM1-EGFP-Top10 recombinant bacterial strain: recombinant plasmid pIRES-TEM1-EGFP is transferred to E.coli In Top10, recombinant bacterial strain pIRES-TEM1-EGFP-Top10 is obtained；

R) it constructs TEM1 positive MS1 cell: rotating into from pIRES-TEM1-EGFP recombinant plasmid electricity is extracted in step q) into TEM1 yin Property cell MS1, screens to obtain TEM1 positive cell line by G418.