CN100335628C - Recombinant calmette-Guerin bacillus vaccine for secretion of human interferon-alphaza and its constructing method - Google Patents
Recombinant calmette-Guerin bacillus vaccine for secretion of human interferon-alphaza and its constructing method Download PDFInfo
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Abstract
The present invention relates to a recombinant Calmette-Guerin bacillus vaccine for the secretion of a human interferon-alpha2a and a constructing method thereof. The method comprises the following steps: a signal peptide segment of a Calmette-Guerin bacillus vaccine Ag85B, which has secretary action, and a gene of human IFN-alpha2a are cloned to pMV261 by a gene engineering technique to obtain a shuttle expression vector of the Calmette-Guerin bacillus vaccine, namely pMSIFN-alpha2a; then, an electroporation technique is adopted to lead the pMSIFN-alpha2a in BCG to construct and recombine a Calmette-Guerin bacillus vaccine rBCGIFN-alpha2a. The copying of the pMSIFN-alpha2a on the BCG, and the secretary action of the signal peptide can effectively secrete IFN-alpha2a. The obtained recombinant Calmette-Guerin bacillus vaccine rBCGIFN-alpha2a of the present invention has the double effects of BCG and IFN-alpha2a, and effectively prevents and treats superficial bladder tumors. The local synergistic action of the recombinant Calmette-Guerin bacillus vaccine rBCGIFN-alpha2a in the urinary bladder cavity is utilized to reduce the dosages of BCG and external IFN-alpha2a, which can overcome the toxic and side effect of BCG therapies.
Description
Technical field
The present invention relates to recombinant bacillus Calmette-Guerin vaccine and the construction process thereof of a kind of secretion of human interferon-α 2a, be specifically related to a kind of recombinant bacillus Calmette-Guerin vaccine and construction process thereof that comprises the main secretion antigen Ag85B of bacille Calmette-Guerin vaccine signal peptide fragment and interferon-' alpha ' 2a (IFN-α 2a), the recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a that obtains is used for prevention and treatment superficial bladder tumor, belongs to gene engineering technology field.
Background technology
Since Morales first report in 1976 is used the shallow property of bacille Calmette-Guerin vaccine (BCG) bladder instillation to treat table tumor of bladder, BCG has obtained good result aspect the tumor of bladder preventing and treating as a kind of effective immunological adjuvant, and BCG irrigation of bladder at present has been regarded as one of the most effective assisting therapy of tumor of bladder.Although BCG shows to have good curative effect aspect the shallow property tumor of bladder in prevention and treatment, still have many problems to need to solve.At first be further to strengthen tumor of bladder to the dabbling immunoreactivity of BCG.Domestic and foreign literature shows, after showing shallow property tumor of bladder surgical blanking, even carry out the BCG bladder instillation to treat of standard, still have patient's immune response for want of of 10%-20% and cause tumor recurrence [Gou Xin etc. " bacille Calmette-Guerin vaccine and interleukin II prevention bladder tumor recurrence long-term observation ", the 21st volume the 9th phase (2000) of medical Leader].Next is to reduce its side effect.After the BCG irrigation of bladder, the patient often has urocystitis symptoms such as frequent micturition, urgent urination, odynuria, blood urine, but the majority spontaneous remission, but still have small number of patients severe complications such as contracture of bladder, BCG septicemia can occur.
Foreign study think anti-knurl mechanism and the local cells immunity of BCG, directly the cell killing etc. of cytotoxic effect, cytokine is closely related, wherein the various kinds of cell factors such as IL-2, IFN-γ, TNF-α participate in the immune response and play an important role.In vitro study and experimentation on animals show that BCG combined utilization IFN-α can strengthen the lethal effect to the tumor of bladder cell by efficiently inducing the generation of IFN-γ, TNF-α etc., obviously suppresses growth of tumor and the recurrence rate that reduces tumour.People's combined utilization BCG and IFN-α treatment superficial bladder tumor is also arranged clinically, and prevention tumor of bladder postoperative recurrence.Compare with simple application BCG perfusion therapy, the patient is stronger to the immunoreactivity of combination therapy, and curative effect is more definite.But there is the shortcoming that the transformation period is short, consumption reaches strong toxicity greatly in above-mentioned cytokine, and high medical expense has limited its popularization [" bacille Calmette-Guerin vaccine associating interleukin II is prevented and treated bladder cancer recurrence " the 21st volume the 9th phase (2000) of medical Leader such as Wang Xulei] clinically simultaneously.The expert begins to pay attention to the molecular biology research that BCG changes vaccine carrier into both at home and abroad in recent years, and has obtained tangible progress.At present, recombinant BCG vaccine has been applied to both at home and abroad schistosomiasis japanica, animal experiment lungy and clinical in, and obtained good effect [the Chinese Amphixenosis's magazine of Gan Yan etc. " polysaccharide adjuvant FQ2 is to the preliminary discussion of Schistosoma japonicum rBCG Sj26GST vaccine synergism and mechanism thereof ", 19.2 (2003)].But make up the recombinant bacillus Calmette-Guerin vaccine that to secrete IFN-α 2a by genetic engineering technique, do not appear in the newspapers as yet with the research that improves the tumor of bladder curative effect.
Summary of the invention
The objective of the invention is to deficiency at traditional bacille Calmette-Guerin vaccine, recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a and the construction process thereof of a kind of secretion of human interferon-α 2a are proposed, the recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a that obtains has the double effects of BCG and IFN-α 2a, strengthen the reactivity of tumor of bladder patient by the efficient secretion that promotes IFN-α 2a, can effectively prevent and treat superficial bladder tumor the BCG immunotherapy; Utilize the part synergy of recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a in bladder cavity to reduce the consumption of BCG and external application IFN-α 2a, can overcome the toxic side effect that BCG treats.
For realizing this purpose, the present invention utilizes genetic engineering technique will play the gene clone of secretory bacille Calmette-Guerin vaccine Ag85B signal peptide fragment and humanIFN-2a to pMV261, obtain bacille Calmette-Guerin vaccine shuttle expression carrier pMSIFN-α 2a, adopt electric transformation technology that pMSIFN-α 2a is imported BCG then and make up recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a, the Ag85B signal peptide is that BCG self plays secretory antigen, can assist the foreign gene secretion to the extracellular.Rely on duplicate the secretion with signal peptide of pMSIFN-α 2a, can efficiently secrete IFN-α 2a at BCG.
The construction process of the recombinant bacillus Calmette-Guerin vaccine rBCG IFN-α 2a of secretion of human interferon of the present invention-α 2a is realized by following step:
Step 1: the structure of bacille Calmette-Guerin vaccine shuttle expression carrier and evaluation
Gene order according to the existing bacille Calmette-Guerin vaccine of GeneBank main secretion antigen Ag85B signal peptide and humanIFN-2a, design the gene fragment of coding Ag85B signal peptide and the amplimer of IFN-α 2a respectively, the upstream and downstream restriction enzyme site of the gene fragment of coding Ag85B signal peptide is respectively BamHI and EcoRI, and the upstream and downstream restriction enzyme site of IFN-α 2a is respectively EcoRI and SalI.(PCR) synthetic respectively obtain the encoding gene fragment of Ag85B signal peptide and gene fragment of IFN-α 2a through the polymerase chain reaction, the gene fragment clone of Ag85B signal peptide of will encoding earlier obtains pMS in the pMV261 carrier, again the gene fragment of IFN-α 2a gene fragment with pMS vector encoded Ag85B signal peptide is connected, obtains bacille Calmette-Guerin vaccine shuttle expression carrier pMSIFN-α 2a.
Step 2: the cultivation of bacille Calmette-Guerin vaccine
Choosing bacille Calmette-Guerin vaccine BCG is Shanghai Denmark strain (H
37Rv56209), provide by the Shanghai institute of Biological Products.BCG dedicated liquid substratum is the Middlebrook7H9 broth (Difco product) that contains 10% nutrition agent ADC (albumin-extrose-catalase), 0.5% tween 80 and 0.2% glycerine.Bacille Calmette-Guerin vaccine is placed on carries out shaking table in the flask that contains liquid nutrient medium and cultivate, shaking table is cultivated 100 rev/mins, three week the back bacille Calmette-Guerin vaccines be in logarithmic phase (OD
600Value=0.6), make the competence bacille Calmette-Guerin vaccine.Can also add penicillin 100 μ g/ml, cycloheximide 100 μ g/ml in the used liquid nutrient medium of the present invention and prevent infectation of bacteria.
Step 3: electric transformation technology makes up recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a
After preparing the competence bacille Calmette-Guerin vaccine, mixing 400 μ l contain 1 * 10 in a centrifuge tube
8The competence bacille Calmette-Guerin vaccine of cfu and 2 μ l contain the shuttle expression carrier pMSIFN-α 2a of 0.4 μ g, place the gene pulse instrument to carry out the electricity conversion and obtain positive recombinant, and electricity changes parameter: voltage is 2500V, and electric capacity is 25 μ F, and resistance is 1000 Ω.Positive recombinant screens in the solid medium that contains 50 μ g/ml kantlex earlier, obtains positive colony rBCGIFN-α 2a after cultivating for two weeks, is seeded in the liquid nutrient medium shaking table growth that contains 50 μ g/ml kantlex then, a large amount of cultivation.
Electricity transforms structure rBCGIFN-α 2a and adopts the solid medium Middlebrook 7H10 (Difco product) that contains 50 μ g/ml kantlex to carry out resistance screening, and liquid nutrient medium is the Middlebrook 7H9 broth that contains 50 μ g/ml kantlex.Solid medium and liquid nutrient medium also can add penicillin 100 μ g/ml, cycloheximide 100 μ g/ml and prevent infectation of bacteria.
Step 4: evaluation and the Function detection of recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a
Carry out acid-fast stain and determine that tentatively rBCGIFN-α 2a is an acid-fast bacilli, extracting rBCGIFN-α 2a plasmid DNA, pcr amplification obtains the gene fragment of IFN-α 2a, Western blotting detects in the rBCGIFN-α 2a culture supernatant He in the thalline the proteic expression of IFN-α 2a, and the ELISA method detects high-load IFN-α 2a in the rBCGIFN-α 2a culture supernatant.
Agents useful for same of the present invention is commercial, and institute of the present invention electricity consumption method for transformation and detection method are conventional currently known methods.
The recombinant bacillus Calmette-Guerin vaccine rBCG IFN-α 2a that the present invention makes up and traditional bacille Calmette-Guerin vaccine are relatively adopting same dose (10
6Cfu/0.1ml) under the situation, can be at external obvious inhibition tumor of bladder cell, there was a significant difference.Be applied to 8 T739 mouse tumor of bladder models (the tumor of bladder cell seeding that comes from this strain forms tumour in the mouse femoribus internus) respectively, adopt the method for locally injected into tumor treatment, find that recombinant bacillus Calmette-Guerin vaccine reaches aspect the immunizing power of this tumour cell in mouse number (8/8 vs.2/8), tumor regression time (1W vs.3W), the no knurl survival time (8W vs.2W) of tumor regression, all is better than traditional bacille Calmette-Guerin vaccine.
The rBCG IFN-α 2a that the present invention makes up can be applied to clinical, and shallow property tumor of bladder is shown in prevention and treatment, not only can improve the curative effect of medication of traditional B CG, reduces the risk of patient tumors recurrence; And can reduce tumor of bladder patient's medical expense, and both helped the patient, also help alleviating social medical expense burden, thereby excellent generalization values and clinical application trend are arranged.
Description of drawings
Fig. 1 is the segmental pcr amplification electrophorogram of the main secretion antigen Ag85B of bacille Calmette-Guerin vaccine signal peptide.
Fig. 2 is the segmental sequencer map of the main secretion antigen Ag85B of bacille Calmette-Guerin vaccine signal peptide.
Fig. 3 is the pcr amplification electrophorogram of humanIFN-2a.
Fig. 4 is the sequencer map of humanIFN-2a.
The behave restriction enzyme digestion and electrophoresis figure of bacille Calmette-Guerin vaccine shuttle expression plasmid pMSIFN-α 2a of Fig. 5.
The behave sequencer map of bacille Calmette-Guerin vaccine shuttle expression plasmid pMSIFN-α 2a of Fig. 6.
Fig. 7 is the acid-fast stain figure of rBCGIFN-α 2a.
Fig. 8 is the pcr amplification electrophorogram of rBCGIFN-α 2a extracting plasmid DNA.
Fig. 9 is the SDS-PAGE result of rBCGIFN-α 2a.
Figure 10 is the Western Blotting result of rBCGIFN-α 2a.
Figure 11 detects the secretion result of rBCGIFN-α 2a for ELISA.
Embodiment
Below in conjunction with drawings and Examples technical scheme of the present invention is further described.
The structure of step 1 bacille Calmette-Guerin vaccine shuttle expression carrier
1. synthetic amplimer
According to the gene order of the existing bacille Calmette-Guerin vaccine of GeneBank main secretion antigen Ag85B signal peptide and humanIFN-2a, the amplimer of design Ag85B signal peptide and IFN-α 2a is respectively
Ag85B?sense：5′-GGATCCATGACAGACGTGAG-3′
antisense：5′-CTGCCGTGCCGATCATCAAT-3′
IFN-α2a?sense：5′-AGCGAATTCTGTGATCTGCCTCAAACCCA-3′
antisense：5′-AGCGTCGACTCATTCCTTACTTCTTAAACTTTCTTGC-3′
Sequencing primer on the pMV261 carrier: 5 '-GACAACTTGAGCCGTCCGTC-3 '
2.Ag85B signal peptide gene is loaded in the pMV261 carrier
Composite signal peptide gene also is cloned into it in pUCm-T carrier
Connect (TAKARA) system: pUCm-T carrier 1 μ l
Signal peptide fragment (about 50ng/ μ l) 4 μ l
Solμtion?I 5μl
16 ℃, connection is spent the night.
Next day, step was as follows with pUCm-TAg85B plasmid transformed competence colibacillus bacterium DH5a:
(1). take out frozen DH5a (200 μ l/Tube), dissolve in the ice;
(2). get 5 μ l and connect liquid and add in the bacterium liquid, mixing gently, ice bath 30min;
(3) .42 ℃ of heat-shocked 1min 30sec, ice bath 2min;
(4). add 500 μ l LB liquid nutrient mediums, cultivate 100rpm * 45min in 37 ℃ of shaking tables;
(5). get 200 μ l and be applied to (100 μ g/ml) on the culture plate that contains ammonia benzyl resistance;
(6) be inverted overnight incubation for .37 ℃, grow to bacterium colony;
(7). the picking mono-clonal contains to 3ml in the LB substratum of ammonia benzyl resistance, cultivates the 250rpm overnight incubation in 37 ℃ of shaking tables.
Extracting pUCm-TAg85B plasmid DNA
Select the bacterium colony that does not have sudden change, the extracting plasmid.As follows according to plasmid extraction test kit experimental procedure:
(1). the 2ml bacterium liquid of overnight incubation is moved in the 2ml centrifuge tube, and high speed centrifugation 30sec abandons most supernatant;
(2). add 250 μ l Solutionl liquid in bacterial precipitation, vibration is to thoroughly suspending;
(3). add 250 μ l Solution, 2 solution, leniently spin upside down centrifuge tube 5-10 time immediately with mixing, make the abundant cracking of thalline until forming bright solution, this step should be finished in 5min;
(4). add Solution 3 solution of 4 ℃ of precoolings of 400 μ l, gentle immediately and centrifuge tube 5-10 time of overturning fully is with mixing, and room temperature leaves standstill 2min, high speed centrifugation 10min;
(5). supernatant is poured in the new centrifuge tube into once more high speed centrifugation 10min;
(6). carefully draw supernatant and transfer to (adsorption column places the waste collection pipe) in the DNA adsorption column, centrifugal 1min abandons filtrate, and adsorption column is placed same collection tube;
(7). add 500 μ l W1 liquid in adsorption column, centrifugal 30sec abandons filtrate, and adsorption column is placed same collection tube;
(8). add 700 μ l W2 liquid in adsorption column, centrifugal 30sec abandons filtrate, and adsorption column is placed same collection tube, repeats once;
(9). high speed centrifugation 1min;
(10). adsorption column is moved in the new clean 1.5ml centrifuge tube, add 60 μ l in DNA adsorption film central authorities and be preheating to 60 ℃ deionized water, room temperature leaves standstill 1min, high speed centrifugation 1min, what obtain is plasmid DNA solution.
Pcr amplification and electrophoresis:
With the extracting plasmid DNA is template, carries out pcr amplification with synthetic signal peptide two ends primer.
Reaction system: deionized water 40.5 μ l
buffer 5μl
dNTPs 1μl
Primer?sig-F 1μl
Primer?sig-R 1μl
Template 1μl
Taq enzyme 0.5 μ l
Reaction conditions: 94 ℃ of 3min
94℃ 30sec
54℃ 1min
72℃ 30sec 30cycles
72℃ 10min
Fig. 1 shows as a result: 2-5 is the pcr amplification band, relatively shows about 120bp with the molecular mass mark, and is identical with the amplified fragments size of gained in theory.
To the evaluation of checking order of extractive pUCm-TAg85B plasmid DNA
Sequencing result is seen Fig. 2, and by NCBI website BLAST contrast, base sequence and Ag85B signal peptide gene are in full accord.Show that the Ag85B signal peptide gene that pcr amplification obtains is entirely true.
Signal peptide gene is cloned in the pMV261 carrier
(1). enzyme T carrier and the pMV261 carrier enzyme of cutting the signal peptide gene place cut system respectively:
buffer 2μl buffer 2μl
pUCm-TAg85B 15μl pMV261 10μl
EcoRI 0.5μl EcoRI 0.5μl
BamHI 0.5μl BamHI 0.5μl
37 ℃ of effect 2hr, 1% agarose gel electrophoresis.
(2). the pMV261 carrier after reclaiming the signal peptide fragment and cutting (Shen friend's gel reclaims test kit), step is as follows:
1.. downcut the sepharose that contains target DNA at ultraviolet lamp, exhaust the liquid of gel surface and shred calculated for gel weight with paper handkerchief;
2.. add the DE-A solution of three gel volumes, behind the mixing in 75 ℃ of heating, during mixed several times, dissolve (about 5min) fully until gel piece;
3.. add the DE-B solution of 1/2 DE-A volume, evenly mixed;
4.. draw the mixed liquid in the above-mentioned steps, transfer to DNA preparation pipe (placing the 2ml centrifuge tube), centrifugal 3600rpm * 1min abandons filtrate;
5.. will prepare pipe and put back centrifuge tube, and add 500 μ l W1 solution, centrifugal 3600rpm * 30sec abandons filtrate;
6.. will prepare pipe and put back centrifuge tube, and add 700 μ l W2 solution, centrifugal 3600rpm * 30sec abandons filtrate; Use the W2 solution washing more once with same method;
7.. will prepare pipe and put back in the centrifuge tube, high speed centrifugation 1min abandons W2 solution only;
8.. will prepare pipe and place clean 1.5ml centrifuge tube, and prepare the film centre at DNA and add 25 μ l and be preheating to 60 ℃ water, room temperature leaves standstill 1min, and high speed centrifugation 1min obtains dna solution.
(3). connect signal peptide and pMV261
Linked system: connect buffer 1 μ l
Signal peptide fragment 5.5 μ l
Ligase enzyme 0.5 μ l
16 ℃ of connections are spent the night, and obtain containing the plasmid pMS of signal peptide gene.
(4). transform (change commentaries on classics, step is the same, and the resistance of bacterium liquid is a kantlex)
3. goal gene IFN-α 2a electrophoresis is identified and dna sequencing
With extractive plasmid DNA is template, carries out pcr amplification with the two ends primer of synthetic IFN-α 2a gene.
Reaction system: deionized water 40.5 μ l
buffer 5μl
dNTPs 1μl
PrimerF 1μl
PrimerR 1μl
Template 1μl
Taq enzyme 0.5 μ l
Reaction conditions: 94 ℃ of 3min
94℃ 30sec
54℃ 1min
72℃ 30sec 35cycles
72℃ 10min
1% agarose gel electrophoresis, DNAMarker confirms.Fig. 3 shows as a result: IL is the pcr amplification band, relatively shows about 495bp with the molecular mass mark, and is identical with the amplified fragments size of gained in theory.
Dna sequencing checking goal gene IFN-α 2a
The PCR product of IFN-α 2a is connected to (step is cloned into the pUCm-T carrier with signal peptide) in the pUCm-T carrier, obtain plasmid pUCm-TIFN-α 2a, transformed competence colibacillus bacterium DH5a, respectively after the extracting plasmid DNA, carry out dna sequencing, result such as Fig. 4, by NCBI website BLAST contrast, base sequence and IFN-α 2a gene are in full accord.Show that the target gene fragment that pcr amplification obtains is entirely true.
Goal gene IFN-α 2a full-length cDNA is loaded among the shuttle plasmid pMS
Reclaim goal gene IFN-α 2a fragment
Enzyme is cut pMS and pUCm-TIFN-α 2a
Enzyme is cut system:
water 2μl
buffer 2μl
pUCm-TIFN-α2a 10μl
EcoRI 0.5μl
SalI 0.5μl
37 ℃ of effect 2hr, 1% agarose gel electrophoresis, DNAMarker confirm that clip size is respectively 4602bp and 495bp.Reclaim pMS carrier, IFN-α 2a fragment.
Ligation
With pMS carrier and IFN-α 2a fragment external connection of row, obtain shuttle expression plasmid pMSIFN-α 2a respectively
Linked system: connect buffer 1 μ l
Exogenous genetic fragment 6.5 μ l
Ligase enzyme 0.5 μ l
Enzyme is cut the checking shuttle expression plasmid
Enzyme is cut system:
water 2μl
buffer 2μl
Shuttle expression plasmid 15 μ l
EcoRI 0.5μl
SalI 0.5μl
37 ℃ of effect 2hr, 1% agarose gel electrophoresis, DNA Marker confirms.The results are shown in Figure 5 shows: IL is that pMSIFN-α 2a is two bands of 4.6kb, 495bp with EcoRI, SalI double digestion result, same expected results.
The shuttle expression plasmid that sequence verification makes up
With the sequencing primer on the pMV261 carrier extractive shuttle expression plasmid pMSIFN-α 2a is carried out sequence verification.Sequencing result is seen Fig. 6, by NCBI website BLAST contrast, and base sequence and IFN-α 2a gene+the Ag85B signal peptide gene is in full accord.
Step 2: the cultivation of bacille Calmette-Guerin vaccine
Choosing bacille Calmette-Guerin vaccine BCG is Shanghai Denmark strain (H
37Rv56209), provide by the Shanghai institute of Biological Products.
BCG dedicated liquid substratum is the Middlebrook7H9 broth (Difco product) that contains 10% nutrition agent ADC (albumin-extrose-catalase), 0.5% tween 80 and 0.2% glycerine.Bacille Calmette-Guerin vaccine is placed on carries out shaking table in the flask that contains liquid nutrient medium and cultivate, shaking table is cultivated 100 rev/mins, three week the back bacille Calmette-Guerin vaccines be in logarithmic phase (OD
600Value=0.6), prepare the competence bacille Calmette-Guerin vaccine according to the following step:
◆ BCG cultivates (rotating speed is 100-120rpm) at 37 ℃ of shaking tables of Middlebrook 7H9 substratum
◆ treat nutrient solution OD
600Be worth at about 0.6 o'clock with in the BCG bacterium liquid adding centrifuge tube, ice bath 2hr;
◆ BCG bacterium liquid is added in the centrifuge tube precooling 2hr in ice chest;
◆ 4 ℃ are centrifugal: 8000g/min * 15min abandons supernatant; With initial volume 1/10, the concentration of precooling is the resuspended thalline of 10% glycerine;
◆ repeat aforesaid operations totally five times;
◆ abandon supernatant, the resuspended thalline of 10% glycerine that adds initial volume 1/50 promptly obtains the competence bacterium, places under the room temperature standby.
Can also add penicillin 100 μ g/ml, cycloheximide 100 μ g/ml in the used liquid nutrient medium of the present invention and prevent infectation of bacteria.
The electric transformation technology of step 3 makes up recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a
◆ mixing 400 μ l competence BCG (1 * 10 in a centrifuge tube
8Cfu) and 2 μ l shuttle expression plasmids (0.4 μ g);
◆ behind the ice bath 10min, the electric shock that changes the 0.2CM of precooling over to transforms in the cup ice bath 10min again, places the gene pulse instrument to carry out electricity and transforms, electricity commentaries on classics V parameter oltage:2500V, Capacitance:25 μ F, Resistance:1000 Ω;
◆ be respectively 22.6,25,26 milliseconds the action time of plasmid pMSIFN-α 2a, pMV261 and simple BCG;
◆ after the electricity commentaries on classics is finished, add 1000 μ l substratum (MADC2Tw) rapidly, cultivate 3hr down for 37 ℃;
◆ get 100-200 μ l bacterium liquid and be applied in the solid medium that contains 50 μ g/ml kantlex, the cultivation that faces up earlier, the plate that overturns behind the 3hr continues to cultivate until finding the clone;
◆ select positive colony after two weeks, shaking table growth in the test tube that contains the 2-3ml liquid nutrient medium.
▲ annotating: competence BCG respectively does a positive control pBCG (only containing blank plasmid pMV261 among the BCG) and negative control (BCG) when transforming herein.
Evaluation and the Function detection of step 4 recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a
1.rBCGIFN-the Z-N method acid-fast stain (preliminary evaluation) of α 2a.
The results are shown in Figure 7: dyeing back opticmicroscope microscopy (eyepiece 10 *, oily mirror 100 *).Under light blue background, acid-fast bacilli takes on a red color.
2.rBCGIFN-α 2a dna level is identified (pcr amplification, electrophoresis are identified)
The extraction of rBCGIFN-α 2a plasmid DNA
◆ the extraction of rBCG plasmid DNA: the rBCG around will cultivating in Erlenmeyer flask divides bottle to the 15ml test tube, makes OD again after shaking table in the test tube cultivated for 2 weeks
600Be about 1, add before the results glycine to 20mg/ml to disturb the synthetic of cell wall, collect thalline after continuing to cultivate certain hour;
◆ after washing twice with physiological saline, thalline is suspended in lysate (2mg/ml N,O-Diacetylmuramidase, 0.3mol/L sucrose, 25mmol/L Tris-Cl (pH=8.0), 25mmol/L EDTA (pH=8.0), after 0.2mg/ml temperature is bathed 24-48hr tetrabromo-mcresolsulfonphthalein), add alkaline SDS (20mg/mlSDS, 3mol/LNaOH);
◆ 37 ℃ of water-bath 12-48hr are limpid until liquid, add the NaAc (pH=4.8) of 3mol/L;
◆ 40 ℃ of water-bath 12hr, centrifugal collection supernatant;
◆ add the Nuclei Lysis Solution of 600 μ l precoolings in the dissolved thalline, 10sec slightly vibrates;
◆ add 3 μ lRNase Solution and mix.Place 15-30min for 37 ℃;
◆ add 200 μ lProtein Precipitation Solution and mix, place 5min on ice;
◆ centrifugal 13000rpm * 4min;
◆ supernatant is changed in the Virahol that contains 600 μ l, mix up and down, centrifugal 13000rpm * 1min;
◆ abandon supernatant, with 70% washing with alcohol once, centrifugal 13000rpm * 1min;
◆ the careful suction abandoned supernatant, drying at room temperature;
◆ add the TE dissolving DNA.
Pcr amplification reaction and electrophoresis are identified:
The condition of pcr amplification reaction, method are with step 1.Electrophoresis qualification result Fig. 8: 1-8 is the pcr amplification band, relatively shows about 495bp with the molecular mass mark, and is identical with the amplified fragments size of gained in theory.
3.rBCGIFN-α 2a protein level is identified
Be cultured to OD in the liquid medium within
600Value is about at 1 o'clock, induce earlier two kinds of rBCG secreting, expressing cytokines (45 ℃, 30min), 4000g/min * 10min, centrifugal 2 times, behind results thalline and the supernatant, thalline with the fragmentation of ultrasonic degradation method, adds lysate (2%SDS earlier again, 0.1M DT, 0.01% tetrabromo-mcresolsulfonphthalein, 0.06M Tris-Cl, 10% glycerine) boil 5min and extract albumen.Respectively sample is washed three times with 1 * PBS, remove the albumin in the nutrient solution.Collect first pass washings and final precipitation and carry out western blot evaluation.
SDS-PAGE separates rBCGIFN-α 2a total protein
Washings and 2 * SDS sample-loading buffer were mixed (10 μ l+10 μ l) by 1: 1, directly go up sample in the boiling water behind the sex change 10min.
Preparation gel slab: press the separation gel of following condition preparation 12% and 5% concentrated glue:
12% separation gel (6ml) 5% concentrates glue (3ml)
30% acrylamide soln 2.4ml 0.5ml
1.5mmol/L?Tris-HClPH8.8 1.55ml /
0.5mmol/L?Tris-HClPH6.8 / 0.38ml
Deionized water 1.93ml 2.1ml
10％SDS 60μl 30μl
10％APS 60μl 30μl
TEMED 2.4μl 3μl
Electrophoresis: concentrate glue 8V/cm, separation gel 15V/cm, constant voltage electrophoresis 2hr treats to finish when tetrabromophenol sulfonphthalein arrives the bottom.Dyeing is observed.The results are shown in Figure 9 shows: swimming lane 1 is shown as the proteic band of IFN-α 2a (the about 19KD of relative molecular mass), and swimming lane 2 is shown as the proteic band of IL-2 (about 15.5KD), and swimming lane 3,4 is not seen protein band in the corresponding position.
Western blotting detects IFN-α 2a protein expression
◆ conventional protein electrophoresis sample separation;
◆ pvdf membrane soaks with methyl alcohol, deionized water rinsing is transferred to balance 20min among the Transferring buffer again, and running gel is put balance 20min among the Transferring buffer, cut out 4 Whatman 3mm filter paper onesize, put balance among the Transferring buffer with glue;
◆ install blot in the following order: negative pole-2 metafiltration paper-running gel-pvdf membrane-2 metafiltration paper-positive pole, in this step, note on the PAGE glue whether dye Marker in advance clear, unclear for fear of the Marker on film after the commentaries on classics film, can be in this step, when glue and film stacked, the Marker on the corresponding glue did mark with ballpoint pen in the corresponding position of film;
◆ 5% skimmed milk/PBST 20ml, horizontal shaking table, room temperature sealing 1hr; Also can first room temperature seal the some time, 4 ℃ are spent the night;
◆ anti-with PBST dilution one, 5%BSA, extension rate: 1000 times, film is enclosed in the plastics bag, do not stay bubble, be fixed on the little shaking table that can vertically rotate deflection angle 70-80 degree, horizontal shaking table, room temperature 1hr with adhesive tape; Wash momently 2 times with PBST earlier, use again＞4ml/cm
2The PBST of (membrane area), room temperature is washed 15min;
◆ anti-with PBST dilution two, 5000 times or 10000 times of dilution 5%BSA, horizontal shaking table, room temperature 1hr; Wash momently 2 times with PBST earlier, use again＞4ml/cm
2The PBST of (membrane area), room temperature is washed 15min;
◆ detection reagent (ECL plus) from 4 ℃ of taking-ups, is being opened forward horizontal stand to room temperature, and the A liquid of detection reagent and B liquid were with 40: 1 mixed, and the consumption of detection reagent is 0.1ml/cm
2
◆ film places on the thieving paper dry, protein powder up, detection reagent is added on the film, covers whole film surface, room temperature 2min;
◆ pick up pvdf membrane with tweezers, the following corner of film is ridden on the paper handkerchief, blot unnecessary detection reagent;
◆ pvdf membrane seals with preservative film, darkroom exposure, time 10sec.
The results are shown in Figure 10 and show, 1-2 is respectively thalline and the culture supernatant among the rBCGIFN-α 2a, all the IFN-α 2a protein expression of visible 19KD; 3-4 is thalline and the culture supernatant of pBCG, does not see protein expression;
5-6 is thalline and the culture supernatant of BCG, does not see protein expression.
3.rBCGIFN-the mensuration of α 2a autocrine IFN-α 2a
Two kinds of rBCG shaking table in test tube was cultivated OD 10 days
600Value is about at 0.5 o'clock, and in the centrifuge tube of packing into, centrifugal 2000rpm * 10min carefully draws supernatant.Detect the level of IFN-α 2a in the culture supernatant according to the operation steps of ELISA test kit specification sheets.Make X-coordinate with standard substance concentration, the standard substance OD that records
620Value is made ordinate zou, connects the coordinate point drawing standard curve of each standard substance with sweep; OD by sample to be measured
620Value is found concentration on typical curve.The results are shown in Figure 11 and show with rBCGIL-2 and compare, the IFN-α 2a that only detects high expression level in rBCGIFN-α 2a culture supernatant is 324.57pg/ml; And all do not detect the expression of IFN-α 2a in the culture supernatant of pBCG and BCG.
Claims (5)
1, the construction process of the recombinant bacillus Calmette-Guerin vaccine of a kind of secretion of human interferon-α 2a is characterized in that comprising the steps:
1) structure of bacille Calmette-Guerin vaccine shuttle expression carrier: design the gene fragment of coding Ag85B signal peptide and the amplimer of IFN-α 2a respectively, the upstream and downstream restriction enzyme site of the gene fragment of coding Ag85B signal peptide is respectively BamHI and EcoRI, the upstream and downstream restriction enzyme site of IFN-α 2a is respectively EcoRI and SalI, gene fragment and IFN-α 2a gene fragment through polymerase chain reaction difference composite coding Ag85B signal peptide, with the gene fragment clone of coding Ag85B signal peptide in the pMV261 carrier, obtain pMS, again the gene fragment of IFN-α 2a gene fragment with pMS vector encoded Ag85B signal peptide is connected, obtains shuttle expression carrier pMSIFN-α 2a;
2) cultivation of bacille Calmette-Guerin vaccine: choosing bacille Calmette-Guerin vaccine is the strain of Shanghai Denmark, bacille Calmette-Guerin vaccine is placed on carries out shaking table in the flask that contains liquid nutrient medium and cultivate, liquid nutrient medium contains 10% nutrition agent ADC, 0.5% tween 80 and 0.2% glycerine, shaking table is cultivated 100 rev/mins, bacille Calmette-Guerin vaccine is in logarithmic phase, OD after three weeks
600Value=0.6 makes the competence bacille Calmette-Guerin vaccine;
3) electric transformation technology makes up recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a: mixing 400 μ l contain 1 * 10 in a centrifuge tube
8The competence bacille Calmette-Guerin vaccine of cfu and 2 μ l contain the shuttle expression carrier pMSIFN-α 2a of 0.4 μ g, place the gene pulse instrument to carry out the electricity conversion and obtain positive recombinant, electricity changes parameter: voltage is 2500V, electric capacity is 25 μ F, resistance is 1000 Ω, and positive recombinant screens in the solid medium that contains 50 μ g/ml kantlex earlier, obtains positive colony rBCGIFN-α 2a after cultivating for two weeks, be seeded in the liquid nutrient medium shaking table growth that contains 50 μ g/ml kantlex then, a large amount of cultivation;
4) evaluation and the Function detection of recombinant bacillus Calmette-Guerin vaccine rBCGIFN-α 2a: carry out acid-fast stain and determine that tentatively rBCGIFN-α 2a is an acid-fast bacilli, after the extracting rBCGIFN-α 2a plasmid DNA, pcr amplification obtains the gene fragment of IFN-α 2a, Western Blot detects in the rBCGIFN-α 2a culture supernatant He in the thalline the proteic expression of IFN-α 2a, and the ELISA method detects high-load IFN-α 2a in the rBCGIFN-α 2a culture supernatant.
2, according to the construction process of the recombinant bacillus Calmette-Guerin vaccine of the secretion of human interferon-α 2a of claim 1, it is characterized in that the gene fragment of described coding Ag85B signal peptide and the amplimer of IFN-α 2a are respectively:
Ag85B?sense：5′-GGATCCATGACAGACGTGAG-3′
antisense：5′-CTGCCGTGCCGATCATCAAT-3′
IFN-α2a?sense：5′-AGCGAATTCTGTGATCTGCCTCAAACCCA-3′
antisense：5′-AGCGTCGACTCATTCCTTACTTCTTAAACTTTCTTGC-3′。
3, according to the construction process of the recombinant bacillus Calmette-Guerin vaccine of the secretion of human interferon-α 2a of claim 1, it is characterized in that the sequencing primer on the described pMV261 carrier is
5′-GACAACTTGAGCCGTCCGTC-3′。
4, the recombinant bacillus Calmette-Guerin vaccine of secretion of human interferon-α 2a of making up of a kind of method that adopts claim 1 or 2 is characterized in that recombinant bacillus Calmette-Guerin vaccine comprises the gene fragment and the IFN-α 2a gene fragment of coding Ag85B signal peptide.
5, the application of the recombinant bacillus Calmette-Guerin vaccine of a kind of secretion of human interferon of claim 4-α 2a is characterized in that the medicine that is used to prepare prevention and treats people's tumor of bladder.
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| 卡介苗加α－干扰素膀胱灌注预防膀胱癌术后复发的疗效 史明，那万里，中国生物制品学杂志，第16卷第2期 2003 * |
| 卡介苗穿梭表达质粒PMSIL22的构建及鉴定 刘海涛等，上海医学，第27卷第5期 2004 * |
| 重组BCG疫苗的研究进展 程继忠等人，微生物不杂志，第16卷第4期 1996 * |
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