CN102191251A - Method for expressing Cecropin CM4 gene in sf9 insect cells - Google Patents
Method for expressing Cecropin CM4 gene in sf9 insect cells Download PDFInfo
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Abstract
The invention relates to the field of biological genetic engineering and discloses a method for expressing Cecropin CM4 gene in sf9 insect cells. The method comprises the following steps: (1), Cecropin CM4 gene is cloned into pFastBacHT B carriers and thus pFastBacH TB-ABP recombinant plasmids are formed; (2), DH10Bac competent cells from Escherichia coli are transformed by the pFastBacH TB-ABP recombinant plasmids to form Bacmid-ABP recombinant baculovirus shuttle vectors; and (3), sf9 insect cells are transfected by the Bacmid-ABP recombinant baculovirus shuttle vectors and thus recombinant virus for expressing the Cecropin CM4 gene is obtained. Through an insect/ baculovirus expression system established by the invention, a novel method for obtaining a large amount of Cecropin is provided, and work foundations of a further research of antibacterial mechanisms and development of novel drugs are laid.
Description
Technical field
The present invention relates to the biological gene engineering field, relate to a kind of method of in insect cell sf9, expressing antibacterial peptide.
Background technology
Antibacterial peptide is the endocrine small molecule active polypeptide of animal and plant body, generally is made up of 15-45 amino acid, is bringing into play important effect in vegeto-animal immunity system.At present, because antibiotic abuse causes Resistant strain to be on the increase, people drop into very big enthusiasm to the research of antibacterial peptide.
Cultivated silkworm antimicrobial peptide belongs to a member in the Cecropin family, for linear, d-helical peptides, contains 35 amino acid; The antibacterial peptide tool is amphipathic: N end possess hydrophilic property, C end have hydrophobicity, antibacterium, antimycotic and antitumor action simultaneously, and normal eukaryotic cell do not had obvious toxic-side effects.
Antibacterial peptide CM 4 is that Zhang Shuanquan etc. separates from fortunatus (Bombyx mori) (No. 12, No. 1 * Soviet Union of Zhejiang farming) and obtains.It is linear, have α spiral amphipathic cationic peptide, form by 35 amino acid, molecular weight is about 3.8KD, and very strong sterilizing ability is arranged, to bacterium, fungi etc. all have effect and do not contain methionine(Met).The bacterium peptide CM4 that holds that extracts from silkworm chrysalis has more antibioticly and kill and wound the ability of some tumour cell, and does not destroy normal cell.Have the report of in escherichia expression system, pichia yeast expression system, expressing antibacterial peptide CM 4 at present, but the relevant report at the expressed in insect cells antibacterial peptide CM 4 is not arranged so far as yet.
Summary of the invention
The objective of the invention is above-mentioned deficiency, a kind of method of expressing antibacterial peptide in insect cell sf9 is provided at prior art.
Purpose of the present invention can be achieved through the following technical solutions:
In insect cell sf9, express the method for antibacterial peptide, comprise the steps:
1) the antibacterial peptide CM 4 gene clone that two ends is comprised BamHI and HindIII restriction enzyme site respectively goes between the BamHI/HindIII site of pFastBacHT B carrier to obtain recombinant plasmid pFastBac HTB-ABP;
2) recombinant plasmid pFastBac HTB-ABP is transformed the DH10Bac competent escherichia coli cell,, obtain to insert the recombinant baculovirus shuttle vectors Bacmid-ABP of ABP gene with baculovirus shuttle vectors Bacmid reorganization wherein;
3) liposome-mediated recombinant baculovirus shuttle vectors Bacmid-ABP transfection Sf 9 insect cell obtains the recombinant virus of expressing antibacterial peptide;
4) cultivate described recombinant virus, express antibacterial peptide CM 4.
Wherein, the antibacterial peptide CM 4 gene that the described two ends of step 1) comprise BamHI and HindIII restriction enzyme site respectively obtains by the following method: the total RNA with fortunatus (Bombyx mori) (No. 12, No. 1 * Soviet Union of Zhejiang farming) is a template, utilize upstream primer sABP-F:SEQ ID NO.1, downstream primer sABP-R:SEQ ID NO.2 obtains described antibacterial peptide CM 4 gene by the rPCR amplification; Be template with described antibacterial peptide CM 4 gene then, utilize upstream primer pFastBacHTB-F:SEQID NO.3, downstream primer pFastBacHTB-R:SEQ ID NO.4 obtains the antibacterial peptide CM 4 gene that two ends comprise BamHI and HindIII restriction enzyme site respectively by pcr amplification.
Present method also comprises the evaluation of described recombinant baculovirus shuttle vectors Bacmid-ABP.
The preferred PCR of the authentication method of described recombinant baculovirus shuttle vectors Bacmid-ABP identifies: respectively with M13-F:SEQ ID NO.5, M13-R:SEQ ID NO.6 and M13-F:SEQ ID NO.5, pFastBacHTB-R:SEQ ID NO.4 and pFastBacHTB-F:SEQ ID NO.3, M13-R:SEQ ID NO.6 is that primer carries out pcr amplification to recombinant baculovirus shuttle vectors Bacmid-ABP, can amplify corresponding 2471bp, 1876bp, being of 740bp target fragment really contains the recombinant baculovirus shuttle vectors Bacmid-ABP that has integrated goal gene.
The Sf-900II SFM that described recombinant virus substratum is the serum-free antibiotic-free.
Beneficial effect:
The contriver has synthesized the primer of insect cell preference codon with reference to the CM4 sequence, and then CM4 is cloned into the pFastBacHTB carrier, and the further recombinant baculovirus shuttle vectors Bacmid-ABP that obtains to contain the ABP gene, by liposome-mediated transfection Sf 9 insect cell, successfully realized the expression of antibacterial peptide CM 4 gene in insect cell, for a large amount of acquisitions of antibacterial peptide CM 4 provide a new way, for working foundation is laid in the research of further antibiotic mechanism and the exploitation of new drug.
The present invention realized first at expressed in insect cells antibacterial peptide CM 4 gene, and expresses CM4 active effect relatively in pichia spp:
1. in insect, express silkworm antibacterial peptide CM 4, since close with the kind of silkworm, be expected to obtain the CM4 that post-treatment is complete, biological activity is high;
2. need only the baculovirus of the recombinant C M4 that obtains high titre, but the direct infection insect cell is easy to purifying, is easy to the scale operation foreign protein.
Description of drawings
Fig. 1, CM4 gene PCR amplified production;
1：CM4?gene(105bp)，M：Marker?2000。
Fig. 2, BamHI/HindIII double digestion are identified recombinant expression vector pFastBac HTB-ABP;
1:pFastBac HTB-ABP BamHI/HindIII enzyme is cut product, and M:Marker 2000.
Fig. 3, Sal I single endonuclease digestion are identified recombinant expression vector pFastBac HTB-ABP;
1:pFastBac HTB; 2:Sal I single endonuclease digestion pFastBac HTB product; 3:Sal I single endonuclease digestion pFastBac HTB-ABP product; M:Marker 2000.
Fig. 4, PCR identify Bacmid-ABP;
1: with M13-F, M13-R is primer amplification Bacmid, 2: with M13-F, M13-R is primer amplification Bacmid-ABP, 3: with M13-F, pFastBacHTB-R is primer amplification Bacmid, 4: with M13-F, pFastBacHTB-R is primer amplification Bacmid-ABP, 5: with pFastBacHTB-F, M13-R is primer amplification Bacmid, 6 is primer amplification Bacmid-ABP with pFastBacHTB-F, M13-R, and LaneM:Marker 2000.
Fig. 5, normal Sf9 cell.
The Sf9 cell of Fig. 6, Bacmid/ABP transfection.
Fig. 7 is. SDS-PAGE and Western blotting that antibacterial peptide CM 4 is merged in reorganization analyze;
M swimming lane: molecular weight marker; 1 swimming lane: the nutrient solution of Gan Raning not; 2 swimming lanes: the cell culture fluid of infection; 3 swimming lanes: the cell pyrolysis liquid of Gan Raning not; 4 swimming lanes: the cell pyrolysis liquid of infection; 5 swimming lanes: the Western blotting result of merging antibacterial peptide.
Embodiment
Employed in the present invention term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense.
Below in conjunction with concrete preparation embodiment and Application Example, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art.Used primer is all indicated when occurring first, and used thereafter same primers as is all identical with the content of indicating first.
The structure of embodiment 1pFastBac HT B/ABP expression vector
(1) obtain the antibacterial peptide CM 4 gene that two ends comprise BamH I and HindIII restriction enzyme site respectively:
sABP-F：
5’-CGTTGGAAGATCTTCAAGAAGATCGAGAAGGTGGGTCAGAACATCC
-3’(SEQ?ID?NO.1)
sABP-R：
5’-GATGGTAGCAGCCTGACCCACCACAGCCACAGCGGGACCAGCCTTC
-3’(SEQ?ID?NO.2)
Tiltedly black matrix is a complementary portion.
Total RNA with fortunatus (Bombyx mori) (No. 12, No. 1 * Soviet Union of Zhejiang farming) is a template, utilizes upstream primer sABP-F:SEQ ID NO.1, and downstream primer sABP-R:SEQ ID NO.2 obtains described antibacterial peptide CM 4 gene (Fig. 1) by the rPCR amplification.
With previous step amplification the antibacterial peptide CM 4 gene be template, utilize upstream primer pFastBacHTB-F:SEQ ID NO.3, downstream primer pFastBacHTB-R:SEQ ID NO.4 obtains the antibacterial peptide CM 4 gene that two ends comprise BamHI and HindIII restriction enzyme site respectively by pcr amplification.
Pcr amplification (introducing restriction enzyme site and enteropeptidase cleavage site), design of primers is as follows:
pFastBacHTB-F：5′-ATCT
GACGACGACGACAAGCGTTGGAAGATCTTC-3′
(SEQ?ID?NO.3) ↑ ↑
BamHI enteropeptidase cleavage site
pFastBacHTB-R：5′-GCTCGA
TCATTAGTTGATGGTAGCAGCCTG-3’
(SEQ?ID?NO.4) ↑
HindIII
(2) pFastBacHT B carrier is gone in the antibacterial peptide CM 4 gene clone
The antibacterial peptide CM 4 gene and the pFastBacHT B carrier (Jin Sirui bio tech ltd) that utilize BamHI and the hindIII two ends that amplification obtains to step (1) respectively to comprise BamHI and HindIII restriction enzyme site respectively carry out double digestion, obtain antibacterial peptide CM 4 gene purpose fragment and pFastBacHT B carrier linear fragment, connect two fragments, obtain connecting product.
(3) connecting product conversion and recombinant plasmid identifies
Flat board is drawn in DH5 α (Beijing 10,000,000,000 new Science and Technology Ltd.s that the create) inoculation of the frozen preservation of glycerine, be inverted overnight incubation for 37 ℃; The picking mono-clonal to the test tube that 3ml LB is housed, 37 ℃ of 220rpm jolting 12h; Draw 1ml bacterium liquid to the 1.5ml centrifuge tube, 4 ℃ of centrifugal 3min of 12000g abandon supernatant; CaCl with 400 μ l 0.1mol/l precoolings
2Resuspended bacterial sediment, the centrifugal 3min of 12000g abandons supernatant; CaCl with 200 μ l 0.1mol/l precoolings
2Resuspended once more bacterial sediment is put on ice and is spent the night; To connect liquid 20 μ l and all add in the 200 μ l competence bacteriums, put 1h on ice; 42 ℃ of heat-shocked 90sec put 5min in the ice rapidly; The LB nutrient solution that adds 37 ℃ of preheatings of 800 μ l; 37 ℃, 220rpm jolting 1h all coats the LB flat board that contains 100 μ g/mlAmp after centrifugal, is inverted overnight incubation for 37 ℃.Picking list colony inoculation contains 37 ℃ of overnight shakings of LB substratum of 100 μ g/ml Amp to be cultivated, and extracts recombinant plasmid and carries out that the BamHI/HindIII double digestion is identified and the evaluation of Sal I single endonuclease digestion, the results are shown in Figure 2 and Fig. 3.Enzyme is cut the correct plasmid called after recombinant expression vector pFastBac HTB-ABP of checking.
The acquisition of embodiment 2 recombinant baculovirus
PFastBac HTB-ABP is transformed DH10Bac competent escherichia coli cell (Jin Sirui bio tech ltd, wherein contain a baculovirus shuttle vectors and be called for short Bacmid, also have a helper plasmid), to obtain to insert the recombinant baculovirus shuttle vectors Bacmid-ABP of ABP gene.
(1) reorganization pFastBac HT B-ABP transforms and enters competence intestinal bacteria DH10Bac (swivel base)
1) get 5 μ l recombinant expression vector pFastBacHT B-ABP and add among the 100ul competence intestinal bacteria DH10Bac, mixing is put into ice-water bath 30min with centrifuge tube gently.Place 42 ℃ of heat-shocked 45s again, move to rapidly then and place 2min in the frozen water;
2) add the LB liquid nutrient medium 900ul that sterilizes in this pipe, 37 ℃ of shaking table 220r/min shaking culture 4h make it to take place swivel base;
3) cell diluent of 10 times of series of usefulness LB inoculum preparation is with 10
-1, 10
-2And 10
-3, different dilutions is respectively got 100 μ l separate application in containing 50 μ g/ml kantlex, 7 μ g/ml gentamicins, 10 μ g/ml tetracyclines, 100 μ g/ml X-gal, the LB flat board of and 40 μ g/ml IPTG;
4) plate is cultivated 48h in 37 ℃ of inversions, observe the growing state of blue hickie.DH10Bac bacterium behind the generation swivel base is inserted into inactivation because of its LacZ gene, and makes the intestinal bacteria bacterium colony that contains the Bacmid that recombinates become white.
(2) extraction of recombinant baculovirus shuttle vectors Bacmid-ABP
Determining of positive table shape:
1) with the rifle head of 10 μ l, 10 bigger white colonies of separation of picking gently, lines on the LB flat board that contains kantlex, gentamicin, tetracycline, X-gal, IPTG equally 37 ℃ of overnight incubation;
2) if the gained bacterium colony still is hickie, illustrating that the bacterium colony of choosing is really contains the positive colony Bac-ABP that has integrated goal gene;
3) the positive white mono-clonal of picking advances to contain the liquid medium of kantlex, gentamicin, tetracycline 37 ℃ of shaking table 225rpm shaking culture 24h from streak plate again.
The extraction of recombinant baculovirus:
1) this nutrient solution branch is filled in the Eppendorf pipe of 1.5ml the centrifugal 1min precipitation of 14000g thalline;
2) abandon clean supernatant, add 300 μ l solution I (15mM Tris-HCl, pH 8.0,10mM EDTA, 100 μ g/ml RNaseA, filtration sterilization), resuspended precipitation;
3) add 300 μ l solution II (0.2N NaOH, 1%SDS, filtration sterilization), gentleness is put upside down mixing, and room temperature is placed 5min, makes the bacterium cracking;
4) slowly dropwise add the potassium acetate solution of 300 μ l 3M (pH5.5), slightly shake in the adition process, thick white tropina and the genomic dna of one deck slowly occurs, and sample is placed 5-10min on ice, the centrifugal 10min of 14000g;
5) with good another the clean Eppendorf pipe of markers, add 800 μ l Virahols, gently with 4) in the gained supernatant be transferred in the new pipe that has added Virahol, note not being mixed with any white precipitate, centrifuge tube is put upside down mixing for several times, place 5-10min on ice, the centrifugal 15min of 14000g abandons supernatant;
6) respectively once, the centrifugal 5min of 14000g with each washing of 75% and 100% ethanol;
7) abandon supernatant, room temperature air-dry (in Cytology Lab under the aseptic condition) 5-10min must express the shaft-like shuttle vectors virus of the reorganization Bacmid-ABP of antibacterial peptide;
8) be dissolved in 40 μ l, 1 * TE damping fluid (10mmol Tris-HCI, lmmol EDTA, pH8.0), 4 ℃ of preservations.
(3) PCR of the shaft-like shuttle vectors virus of reorganization Bacmid-ABP identifies
M13 primer pairing site among the Bacmid is positioned at the mini-attTn7 both sides, with M13 upstream primer and the shaft-like shuttle vectors virus of M13 downstream primer amplification reorganization Bacmid-ABP, is contrast with wild baculovirus (Jin Sirui bio tech ltd).
M13-F：5′-GTTTTCCCAGTCACGAC-3′(SEQ?ID?NO.5)；
M13-R：5′-CAGGAAACAGCTATGAC-3′(SEQ?ID?NO.6)；
pFastBacHTB-F：5′-ATCT
GACGACGACGACAAGCGTTGGAAGATCTTC-3’；
(SEQ?ID?NO.3)。
pFastBacHTB-R：5′-GCTCGAAAGCTTTCATTAGTTGATGGTAGCAGCCTG-3’(SEQ?IDNO.4)。
Be primer with M13-F, M13-R and M13-F, pFastBacHTB-R and pFastBacHTB-F, M13-R respectively, the shaft-like shuttle vectors virus Bacmid-ABP that recombinates carried out PCR identify, the results are shown in Figure 4.As seen from Figure 4 for the shaft-like shuttle vectors virus of reorganization Bacmid-ABP, with M13-F, M13-R is primer, can amplify the band of 2471bp, with M13-F, pFastBacHTB-R is primer, can amplify the band of 1876bp, with pFastBacHTB-F, M13-R is primer, can amplify the band of 740bp.
The shaft-like shuttle vectors virus of embodiment 3 reorganization Bacmid-ABP transfection Sf9 cell
(1) transfection sf9 monolayer cell
Every hole is with the Sf-900 II SFM nutrient solution inoculation 9 * 10 of the serum-free antibiotic-free of 2ml in six orifice plates of 35mm
5Individual Sf9 cell (Nanjing Jin Sirui bio tech ltd), overnight incubation treats to carry out when cell density grows to 60%-70% transfection.The following solution of preparation in aseptic Eppendorf pipe,
A liquid: 5 μ l reorganization Bacmid-ABP adds the Sf-900 II SFM of 100 μ l serum-free antibiotic-frees;
B liquid: 6 μ l lipofectamine Cellfectin add the Sf-900 II SFM of 100 μ l serum-free antibiotic-frees; Then A liquid and B liquid are mixed gently, room temperature is placed 15-45min.Remove the nutrient solution in each hole in six orifice plates simultaneously,, abandon clean nutrient solution with the Sf-900II SFM of serum-free antibiotic-free washing one time.In above-mentioned mixed solution, add the Sf-900II SFM of 800 μ l serum-free antibiotic-frees,, in each hole of resorb, place 27 ℃ to hatch 5h to the about 1ml of cumulative volume.Remove the DNA mixed solution behind the 5h, every hole adds 2ml Sf-900II SFM, cultivates 72h observation of cell pathology situation afterwards in 27 ℃ of incubators, and Fig. 5 and Fig. 6 are seen in contrast before and after the cytopathy.
(2) acquisition of recombinant baculovirus and expansion
After treating that obvious pathology situation appears in transfected cell, collect in each hole and contain recombinant baculovirus particulate culture supernatant, the centrifugal 5min of 500g removes cell and big fragment, and supernatant is divided into 4 ℃ of preservations of aliquot lucifuge, this be P1 for virus, can be used for infecting once more the Sf9 monolayer cell to obtain.
For obtaining infectious titer, the P1 that available following formula calculates required inoculation when obtaining a certain specific MOI is for virus:
P(k)＝1-P(0)
MOI＝-InP(0)
P (k) is the percentage of infected cell, and P (0) is the percentage of infected cell not,
MOI (multiplicity ofinfection) claims infection multiplicity, is the value of virus and the ratio of cell quantity when infecting.
With MOI is 0.1 to carry out virus amplification, plants 2 * 10 in six orifice plates
6Cells/well, 27 ℃ of cultivations, microscopy cell attachment situation behind the 1h, add an amount of P1 in every hole, in 27 ℃ of incubators, cultivate 48h, microscopy cytopathy situation behind the 48h, from every hole, collect the substratum that 2ml contains virion, 500g is centrifugal, and 5min gets supernatant, and 4 ℃ keep in Dark Place, and this is P2 for virus.Can in kind increase and obtain P3 for virus.
The expression and the evaluation thereof of embodiment 4 target proteins
(1) expression of target protein
1) with 6 * 10
5The Sf9 cell switching of/ml is cultivated, more than the cell attachment 30min;
2) removing the Sf-900 II SFM nutrient solution of serum-free antibiotic-free, add fresh medium, is 10 to carry out protein expression with MOI, adds P3 for recombinant virus 100 μ l in every hole, cultivates in 27 ℃;
3) (Fig. 6) receives cell analysis protein expression situation after obvious pathology appears in cell.
What (2) expression product distributed determines
Receive cell and nutrient solution supernatant, wherein the centrifugal 10min of nutrient solution supernatant 10000g receives supernatant, and cell washes twice with PBS, with cell pyrolysis liquid (RIPA buffer) cracking on ice 0.5 hour, with the cell sleaker cell is scraped 4 ℃ of 10000g centrifuging and taking supernatants again.Get 2 * sample-loading buffer that 10 μ l supernatant liquors add equivalent, precipitation is got 10 μ l with 500 μ l, 1 * sample-loading buffer after resuspended, and constant voltage 100V carries out 12%SDS-PAGE, and coomassie brilliant blue R250 dyeing shows is with, and the results are shown in Figure 7 (swimming lanes 1～4).2 * sample-loading buffer prescription: 1.0M Tris (pH=6.8) 0.48ml, 100% glycerine 2ml, 10%SDS 1.6ml, beta-mercaptoethanol 0.4ml, tetrabromophenol sulfonphthalein 0.02g, water 15.52ml.
(3) evaluation of recombinant antibacterial peptide
Sample thief 10 μ l add 2 * sample-loading buffer that equivalent contains beta-mercaptoethanol, and constant voltage 100V carries out 12%SDS-PAGE, change film, utilize His antibody to detect.
The operating process of Western blotting is as follows:
1) preparation: take out the PAGE glue (Fig. 7, swimming lane 4) that electrophoresis finishes, cut and concentrate glue and following unnecessary part, placed 1XTransfer buffer balance 5 minutes; Simultaneously, film, filter paper and sponge all are soaked among the 1XTransfer buffer, 5 minutes;
2) change film: spreading successively on the transfer box in transfer buffer behind the wetted sponge and filter paper, the separation gel part of tiling SDS-PAGE is put NC film, two layers of filter paper and sponge, the bubble of rushing above more successively; Constant current 250mA then, ice bath changeed film 90 minutes; Change film and finish back taking-up NC film, it is shaken in TBST wash 10min;
3) seal: the NC film is washed away 1h in 20ml blocking buffer after, shake among the 15ml TBST and wash 3 times, each 5min;
4) incubate one anti-: abandon TBST, add 4 ℃ of overnight incubation of an anti-mouse-anti His antibody of confining liquid dilution, TBST shakes and gives a baby a bath on the third day after its birth time, each 5min;
5) incubate two anti-: again with the sheep anti-mouse igg incubated at room 1h of HRP mark, TBST shakes and gives a baby a bath on the third day after its birth time, each 5min;
6) near colour developing: the purpose band,, the results are shown in Figure 7, swimming lane 5 with the TMB colour developing.As seen from Figure 7, a differential protein band is arranged, illustrate that the target protein antibacterial peptide CM 4 obtains expressing, and the expressing protein major part exists all in the cell pyrolysis liquid supernatant at the 7.4kD place.
Claims (5)
1. in insect cell sf9, express the method for antibacterial peptide, it is characterized in that comprising the steps:
(1) the antibacterial peptide CM 4 gene clone that two ends is comprised BamHI and HindIII restriction enzyme site respectively goes between the BamHI/HindIII site of pFastBacHTB carrier to obtain recombinant plasmid pFastBac HTB-ABP;
(2) recombinant plasmid pFastBac HTB-ABP is transformed the DH10Bac competent escherichia coli cell,, obtain to insert the recombinant baculovirus shuttle vectors Bacmid-ABP of ABP gene with baculovirus shuttle vectors Bacmid reorganization wherein;
(3) liposome-mediated recombinant baculovirus shuttle vectors Bacmid-ABP transfection Sf 9 insect cell obtains the recombinant virus of expressing antibacterial peptide;
(4) cultivate described recombinant virus, express antibacterial peptide CM 4.
2. the method for in insect cell sf9, expressing antibacterial peptide according to claim 1, it is characterized in that the antibacterial peptide CM 4 gene that the described two ends of step 1) comprise BamHI and HindIII restriction enzyme site respectively obtains by the following method: the total RNA with No. 12, No. 1, fortunatus Zhejiang farming * Soviet Union is a template, utilize upstream primer sABP-F:SEQ ID NO.1, downstream primer sABP-R:SEQ ID NO.2 obtains described antibacterial peptide CM 4 gene by the rPCR amplification; Be template with described antibacterial peptide CM 4 gene then, utilize upstream primer pFastBacHTB-F:SEQ ID NO.3, downstream primer pFastBacHTB-R:SEQ ID NO.4 obtains the antibacterial peptide CM 4 gene that two ends comprise BamHI and HindIII restriction enzyme site respectively by pcr amplification.
3. the method for expressing antibacterial peptide in insect cell sf9 according to claim 1 is characterized in that present method also comprises the evaluation of described recombinant baculovirus shuttle vectors Bacmid-ABP.
4. the method for in insect cell sf9, expressing antibacterial peptide according to claim 3, the authentication method that it is characterized in that described recombinant baculovirus shuttle vectors Bacmid-ABP is that PCR identifies: respectively with M13-F:SEQ ID NO.5, M13-R:SEQ ID NO.6 and M13-F:SEQ ID NO.5, pFastBacHTB-R:SEQ ID NO.4 and pFastBacHTB-F:SEQ ID NO.3, M13-R:SEQ ID NO.6 is that primer carries out pcr amplification to recombinant baculovirus shuttle vectors Bacmid-ABP, can amplify corresponding 2471bp, 1876bp, being of 740bp target fragment really contains the recombinant baculovirus shuttle vectors Bacmid-ABP that has integrated goal gene.
5. the method for expressing antibacterial peptide in insect cell sf9 according to claim 1 is characterized in that the Sf-900 II SFM that described recombinant virus substratum is the serum-free antibiotic-free.
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| 《南京大学学报》 19960731 谢维等 中国家蚕抗菌肽CMⅣ基因的合成与克隆 474-478 2 第32卷, 第3期 * |
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