CN1140628C - Human interleukin-12 recombinant insect virus strain and its prepn - Google Patents
Human interleukin-12 recombinant insect virus strain and its prepn Download PDFInfo
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Abstract
The present invention discloses a human leukocyte interleukin-12 recombinant insect virus strain and a preparation method thereof. The recombinant insect virus strain is a recombinant autographa californica nuclear polyhedrosis AcNPV-hIL12 virus strain of which the preservation number is CCTCCNO. V200108. A human leukocyte interleukin-12 expression box is inserted into the recombinant virus strain, wherein the expression box comprises a dual-promoter sequence, a P35 subunit DNA coding sequence of the human leukocyte interleukin-12, and a P40 subunit DNA coding sequence; the P35 subunit gene is positioned at the down stream of the polyhederin, and the P40 subunit gene is positioned at the down stream of the P10 promoter. The virus strain can express the human leukocyte interleukin-12 with high efficiency; the cell factor is safe and reliable to a human body and is suitable for treating infectious diseases such as tumors, bacteria, viruses, parasites, etc.
Description
Technical field
The present invention relates to biotechnology, more specifically relate to Ro 24-7472/000-12 recombinant insect virus strain (AcNPV-hIL12), also relate to a kind of preparation method of expressing human interleukin 12 recombinant insect virus strain.
Background technology
(human lnterlukin12 hIL-12) is the important cytokine of human body to Ro 24-7472/000-12, also is referred to as natural kill cell stimulating factor (NKSF) or cytotoxicity lymph maturation factor (CLMF).1989, Kobayashi etc. at first separate and purifying hIL-12 (Kobayashi M, et al.J Exp Med, 1989ml70:827-845).1991, Gubler and Wolf cloned hIL-12 cDNA complete sequence, and expressed (Gubler U, et al, Proc Nati Acad SciUSA, 1991,88:4143-4147 in mammalian cell; Wolf S F, et al, J Immunol, 1991,146:3047-3081).HIL-12 is by not having the glycoprotein heterodimer that two protein subunit P35 and the P40 of gene-correlation link to each other and constitute a 70KDa by disulfide linkage.The P35 subunit has 197 amino-acid residues, and molecular weight is 31KDa; The P40 subunit has 306 amino-acid residues, and molecular weight is 40KDa.The P35 subunit is function subunit (Gubler, at al.J Biol Chem, 1995 of transmission information, 270:11) and the P40 subunit is hIL-12 and cell receptor bonded functional protein (Gillessen S, et al European Journal of Immunology, 1995,25:1).IL-12 mainly is a kind of heterodimer cytokine that is produced by antigen presenting cell, can significantly strengthen the killing activity of NK/LAK cell, promotes the ability of answering that opens of Specific CTL Cells, and inducing T cell and a large amount of secretion of gamma-IFN of NK cell start T
H0 cell is to T
H1 cells whose development will play a significant role in multiple diseases such as antiviral, antitumor, anti-cell bacterial parasite infection curing, acquired immune deficiency syndrome (AIDS).(Lamont?AG，etal，lmmunology?Today，1996，17：214-217)。
Chinese scholars (Yang Lin etc., microorganism journal, 2001,41 (1) 35-41, Wei Haiming etc., Chinese microbiology and Journal of Immunology, 2000,20 (6) 584-587, Kazuaki Takehara et al VeterinaryImmunology and Immunopathology, 2000,77:15-25) utilize the two subunits of insect viruses dual-expression vector coexpression interleukin 12, perhaps express P35, P40 subunit (Shen Hui etc., biotechnology journal respectively, 1998,14 (4) 360-364).Perhaps utilize the E1 and the E3 defect area of adenovirus carrier, respectively with P40 with P35 is cloned into E1 and the E3 defect area is expressed (Zhang Weiping etc., Chinese Medical Journal, 1998,78 (1) 33-36; Chinese patent CN98126748.3, December 13 1998 applying date), and the virus applications that can express hIL-12 in treatment.
Interleukin 12 is in the cytokine of being found at present immunologically competent cell in the body to be induced regulating effect a kind of cytokine strong and the most widest in area, participates in antitumor, the antianaphylaxis of antibody, anti-infective process.At present, the U.S. has been used for the treatment of IL-12 AIDS patient and tumour patient, has now entered the clinical III phase.IL-12 is used for anti-leishmania, antimalarial protozoon, and tuberculosis disease, anti-schistosomiasis have also entered experimentation on animals.1997, Michael Brunda study group report, IL-12 has obvious effect to kinds of tumors, comprises to nephroncus, melanoma Colon tumour and 14 kinds of hypoploidy tumours.IL-12 is being used for the treatment of in the model of mouse 16F16 melanoma, IL-12 is injected in and suffers from the knurl mouse, can suppress tumor growth; Directly IL-12 is expelled to tumor locus and causes tumour regression, after long-term disposal, tumor regression, animal is cured.
Though studies show that hIL-12 great application prospect is arranged, and have the method for many hIL-12 of preparation, the problem of the use amount of hIL-12, excessive use can cause the immune response of body, the toxigenicity effect.Therefore, adopt engineered method to obtain hIL-12 and be used for clinical treatment, timely and appropriate discovery provides patient, is better than gene therapy, and exists the problem of the virus safe of importing in the gene therapy.In addition, although there is the scholar to express P35, P40 subunit respectively, owing to the P35 among the hIL-12, P40 subunit just have activity through forming dimer.Therefore, need the two subunit coexpressions of P35, P40.Chinese scholars (Wei Haiming etc., China microbiology and Journal of Immunology, 2000,20 (6) 584-587) also utilize coexpression P35 of AcNPV system and the two subunits of P40, but P35 subunit cDNA places P10 promotor lower end, and P40 subunit cDNA places Polyhedrin promotor downstream.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can efficiently express Ro 24-7472/000-12 recombinant insect virus strain, the overexpression that this insect virus strain can not form the P40 subunit forms the P40 homodimer.
Another object of the present invention provides the preparation method that a kind of preparation efficiently expresses the recombinant insect virus strain of human interleukin 12, can not form the overexpression formation P40 homodimer of P40 subunit with the recombinant insect virus strain of this method preparation, can efficiently express Ro 24-7472/000-12, and the Ro 24-7472/000-12 pair human body that produces is safe and reliable.
In order to achieve the above object, the present invention is described below:
Reorganization clover crazing moth Nucleopolyhedrovirus one strain called after reorganization AcNPV strain (AcNPV-hIL12 provided by the present invention, CCTCC NO:V200108), the reorganization autographa california nuclear polyhedrosis virus strain of a kind of expressing human interleukin 12 (hIL-12), it is characterized in that, this recombinant virus has inserted Ro 24-7472/000-12 expression cassette, expression cassette comprises the double-promoter sequence, the P35 subunit dna encoding sequence of Ro 24-7472/000-12 and P40 subunit dna encoding sequence, the P35 subunit gene is positioned at Polyhedrin promotor downstream, and the P40 subunit gene is positioned at P10 promoter promotor downstream.
A kind of preparation method who makes up the strain of reorganization autographa california nuclear polyhedrosis virus of the present invention, its step is as follows:
A) branch adds amplification p35 subunit dna fragmentation and p40 subunit dna fragmentation, and is cloned into pCR respectively
2.1 in the carrier, obtain pCR35 and pCR40 plasmid.
B) with Bgl II and Bam HI double digestion pCR40 plasmid, the P40 subunit is cloned in the pAcUW51 plasmid DNA that Bgl II enzyme is cut, obtains the pAcUW51-P40 plasmid;
C) cut plasmid DNA with Eag I enzyme, and the P35 subunit is cloned in the pAcUW51-P40 plasmid that Bam HI enzyme is cut, obtain the pAcUW51-ILl2 plasmid with T4 DNA polymerase Kelnow fragment tack processing pCR35;
D) the linear autographa california nuclear polyhedrosis virus gene DNA of pAcUW51-ILl2 plasmid and deadly defective type cotransfection insect cell Sf9 clone;
E) plaque select;
F) PCR identifies the strain of reorganization autographa california nuclear polyhedrosis virus.
In a preference of the present invention, a kind of method that makes up the strain of reorganization autographa california nuclear polyhedrosis virus is provided, it is characterized in that being that this method comprises:
A) RT-PCR amplification 667bp P35 subunit fragments and 1000bp P40 subunit fragments are cloned into pCR respectively with this two fragment
2.1 in the carrier, connect, transform, screen, identify by enzyme, obtain to contain the bacterial strain of recombinant plasmid pCR35: E.coli DH5 α (pCR35), the bacterial strain that contains recombinant plasmid pCR40 and E.coli DH5 α (pCR40) [obtaining two kinds of bacterial strain E.coli (pCR35) and E.coli (pCR40)];
B) obtain 1000bp P40 subunit fragments by cut the pCR40 plasmid DNA with Bgl II and two kinds of enzymes of Bam HI, this fragment cloning in the pAcUW5l plasmid DNA of cutting through Bgl II enzyme, is connected, transforms, screens, identifies the E.coli DH5 α (pAcUW5l-P40) that obtains to contain recombinant plasmid pAcUW51-P40 through enzyme;
C) handle pCR35 plasmid DNA acquisition P35 subunit fragments by cutting with Eag I enzyme with T4 DNA polymerase Klenow fragment tack, this fragment cloning in the pAcUW5l-P40 plasmid DNA of cutting through Bam HI enzyme, is connected, transforms, screens, identifies the E.coliDH5 α (pAcUW51-ILl2) that obtains to contain recombinant plasmid pAcUW-ILl2 through enzyme;
D) the greedy noctuid Sf9 cell in the linear autographa california nuclear polyhedrosis virus gene DNA of pAcUW51-ILl2 plasmid DNA and deadly defective type cotransfection meadow;
E) plaque select;
F) separate reorganization autographa california nuclear polyhedrosis virus strain ANPV-hILl2, CCTCC-V200108.
In the present invention, the human interleukin 12 encoding sequence comprises the dna encoding sequence of P35 subunit and P40 subunit.
In the present invention, the human interleukin 12 expression cassette is meant that the sequence of dna encoding of the P35 that contains IL-12 and P40 subunit and the sequence of expressing required element form, and comprises promotor.
In the present invention, can provide the insect viruses gene DNA of use to be meant insect nuclear polyhedrosis virus gene DNA, autographa california nuclear polyhedrosis virus gene DNA preferably, more preferably causing death lacks the linear autographa california nuclear polyhedrosis virus gene DNA of dead defective type.
Because the heterodimer that IL-12 is made up of through disulfide linkage two subunits of P35, P40, have only the IL-12 of correct assembling just to have biologic activity, and P35, P40 monomer all can not bring into play the biological action of IL-12, opposite P40 dimer also can play antagonism IL-12, so the two subunit co-expression carriers of IL-12 have more using value.
Use plasmid pAcVW51 structure and contain hIL-12P35 subunit and P40 subunit co-expression carrier, if the P40 subunit is in polyhedrin promotor downstream, then in the expression process, because the Polyhedrin promotor is the promotor stronger than P10, then the P40 expression is excessive, form the P40 homodimer, and the P40 homodimer is the antagonist of IL-12, the overexpression of P40 subunit suppresses to have the formation of bioactive IL-12, therefore, in the present invention, the enzyme butt formula by different places P10 promotor downstream with the P40 subunit, and the P35 subunit is positioned at strong promoter Polyhedrin downstream, construct the insect virus strain of P35 subunit, can efficiently express IL-12 like this, and the IL-12 that expresses is safe and reliable with respect to the two subunit coexpressions of P40 subunit overexpression IL-12.Reorganization AcNPV strain provided by the invention contains two promotors, is respectively P10 and Polyhedrin promotor.In the hIL-12 expression cassette, the P35 subunit gene is positioned at Polyhedrin promotor downstream; The P40 subunit gene is positioned at P10 promotor downstream.This being built with is beneficial to P35 and P40 formation dimer, expresses the formation homodimer and avoid P40 to cross.Utilize this recombinant insect virus can efficiently express Ro 24-7472/000-12, the people Bai Zujie element-12 of purifying is applicable to the treatment of infectious diseases such as tumour, bacterium, virus and parasite.
Description of drawings
Fig. 1 .hIL-12 P35, P40 subunit PCR product are identified
Fig. 2. the structure of recombinant plasmid pCR35
Fig. 3 .pCR35 enzyme is cut with PCR and is identified
Fig. 4. the structure of recombinant plasmid pCR40
Fig. 5 .pCR40 enzyme is cut with PCR and is identified
Fig. 6 .hIL-12P35 dna sequence dna
Fig. 7 .hIL-12 P40 dna sequence dna
Fig. 8. the structure of recombinant plasmid pAcUW51-P40
Fig. 9 .pAcUW51-P40 enzyme is cut with PCR and is identified
Figure 10. the structure of recombinant plasmid pAcUW51-IL12
Figure 11 .pAcUW51-IL12 enzyme is cut with PCR and is identified
Figure 12. recombinant virus Ac-hIL12 PCR identifies
Embodiment
Below in conjunction with accompanying drawing the present invention is described in further detail:
The 1. P35 subunit cDNA of human interleukin 12 and clone and the evaluation of P40 subunit cDNA of embodiment
Collection is through PDBu (100nM, Sigma) stimulate cultivation 20hKB cell, collecting cell adopts guanidinium isothiocyanate single stage method extracted total RNA, the MMLV reversed transcriptive enzyme synthesizes cDNA, is that template adopts following P35 and P40 primer PCR amplification P35 and P40 dna fragmentation respectively with it.P35 primer: P1 TTGATCA ATG TGT CCA GCG CGC AGCCT
P2 AGTGACG AGC TAT CTG AAT GCTTCC TAAP40 primer: P3 AGATCT ATG TGT CAC CAG CAGTTG GT
P4?TGGATCCCTA?AC?TGCAGGG?CAC?AGATG
PCR reaction cumulative volume 50 μ l.Reaction conditions is: 94 ℃ of pre-sex change 5min, the PCR loop parameter: 94 ℃ 60 seconds, 58 ℃ 90 seconds, 72 ℃ 120 seconds, circulate 30 times, circulate in 72 ℃ for 1 time and extend 7min at last.Through the 0.8%Agarose electrophoresis detection, obtain single 667bp P35 cDNA fragment and 1000bp P40cDNA fragment.The 0.8%Agarose gel electrophoresis the results are shown in Figure 1.
Embodiment is recombinant plasmid pCR35 and pCR40 structure and evaluation 2.
(1) recombinant plasmid pCR35 makes up and identifies
The hIL-12 P35 cDNA fragment cloning that 1. embodiment is obtained is to pCR
2.1 in the carrier, connect product Transformed E .coli DH5 α, obtain positive transformant through Ampcillin and the two anti-plate screenings of Kanamycin.This construction recombination plasmid called after pCR35.The pCR35 plasmid construction is seen Fig. 2.Recombinant plasmid pCR35 obtains two dna fragmentations of 0.7kb and 3.8kb through Eag I single endonuclease digestion.PCR35 obtains～dna fragmentation of 4.5kb through Not I single endonuclease digestion.In addition, be primer with P1, P2 primer, be template with pCR35 DNA, pcr amplification obtains dna fragmentation of 0.67kb.PCR reaction cumulative volume 50 μ l, reaction conditions is: 94 ℃ of pre-sex change 5min, the PCR loop parameter: 94 ℃ 60 seconds, 58 ℃ 90 seconds, 72 ℃ 120 seconds, circulate 30 times, circulate in 72 ℃ of extension 7min last 1 time.The gel electrophoresis result conforms to expection, sees Fig. 3.
(2) recombinant plasmid pCR40 makes up and identifies
1. embodiment is obtained hIL-12 P40 cDNA fragment cloning to pCR
2.1 in the carrier, connect product Transformed E .coli DH5 α, obtain positive transformant through Ampcillin and the two anti-plate screenings of Kanamycin.The recombinant plasmid called after pCR40 of this structure.The pCR40 plasmid construction is seen Fig. 4.Recombinant plasmid pCR40 obtains two dna fragmentations of 1.0kb and 3.9kb through Bgl II and Bam HI double digestion.PCR40 obtains～dna fragmentation of 4.9kb through Not I single endonuclease digestion.In addition, be primer with P3, P4, be template with pCR40 DNA, pcr amplification obtains dna fragmentation of 1.0kb.PCR reaction cumulative volume 50 μ l, reaction conditions is: 94 ℃ of pre-sex change 5min, the PCR loop parameter: 94 ℃ 60 seconds, 58 ℃ 90 seconds, 72 ℃ 120 seconds, circulate 30 times, circulate in 72 ℃ of extension 7min last 1 time.The gel electrophoresis result conforms to expection, sees Fig. 5.
Embodiment is hIL-12 P35 and P40 sequencing 3.
2. embodiment is obtained pCR35 and pCR40 analyzes through full-automatic order-checking (ABI 373, PE company), sequencing result with report consistent (Wolf, S.F, et al.1991, J.lmmunol.146:3074-3081), P35, P40DNA sequence are seen Fig. 6, Fig. 7 respectively.
Embodiment is structure and the evaluation of recombinant plasmid pAcUW51-P40 4.
Plasmid pCR40 DNA with 2. Bgl II and Bam HI double digestion embodiment obtain obtains 1.0kb and 3.9kb dna fragmentation.With Bgl II single endonuclease digestion plasmid pAcUW51 DNA, obtain the 5.9kb dna fragmentation.Adopt 0.8% low melting-point agarose gel electrophoresis, reclaim 1.0kb and 5.9kb dna fragmentation.Connect with the T4 dna ligase, connect product Transformed E .coli DH5 α, obtain positive transformant through the Amp plate screening.The recombinant plasmid called after pAcUW51-P40 of this structure sees Fig. 8.Recombinant plasmid pAcUW51-P40 obtains dna fragmentation of 6.86kb through Bgl II single endonuclease digestion.Dna fragmentation of 6.86kb appears in Bam HI single endonuclease digestion.PAcUW51-P40 is through Eco RI single endonuclease digestion, and forward connects and 0.26kb and two dna fragmentations of 6.61kb occur; Oppositely connect and 6.11kb and two dna fragmentations of 0.76kb occur.In addition, P3, P4 are primer, are template with pAcUW51-P40 DNA, and pcr amplification obtains dna fragmentation of 1.0kb.PCR reaction cumulative volume 50 μ l, reaction conditions is: 94 ℃ of pre-sex change 5min, the PCR loop parameter: 94 ℃ 60 seconds, 58 ℃ 90 seconds, 72 ℃ 120 seconds, circulate 30 times, circulate in 72 ℃ of extension 7min last 1 time.Gel electrophoresis the results are shown in Figure 9.
Embodiment is the structure of recombinant plasmid pAcUW51-IL12 5.
With Eag I single endonuclease digestion and with the processing of T4 archaeal dna polymerase Klenow fragment tack 2. embodiment is obtained plasmid pCR35 DNA, obtain 0.7kb and two dna fragmentations of 3.8kb.With the BamHI single endonuclease digestion and with the processing of T4 archaeal dna polymerase Klenow fragment tack 4. embodiment is obtained plasmid pAcUW51-P40DNA, obtain the 6.86kb dna fragmentation.Adopt 0.8% low melting-point agarose gel electrophoresis, reclaim 0.7kb P35cDNA fragment and 6.86kb dna fragmentation.Connect with the T4 dna ligase, connect product Transformed E .coli DH5 α, obtain positive transformant through the Amp plate screening.The recombinant plasmid called after pAcUW51-IL12 of this structure sees Figure 10.Recombinant plasmid pAcUW51-IL12 obtains the 7.59kb dna fragmentation through Bgl II single endonuclease digestion.PAcUW51-IL12 is through the EcoRI single endonuclease digestion, and forward connects and 0.26kb, 1.77kb and three dna fragmentations of 5.57kb occur; Oppositely connect and 0.26kb, 1.18kb and three dna fragmentations of 6.16kb occur.In addition, be primer with P1, P2, be template with pAcUW51-IL12 DNA, pcr amplification obtains 667bp P35cDNA fragment; P3, P4 are primer, are template with pAcUW51-IL12 DNA, and pcr amplification obtains 1000bp P40 cNDA fragment.Gel electrophoresis the results are shown in Figure 11.
6. embodiment expresses the structure of hIL-12 recombinant baculovirus AcNPV-hIL12
5. embodiment is obtained pAcUW51-IL12 DNA and BaculoGold
TMThe greedy frugiperda cell Sf9 inoculation 2 * 10 in Linearized BaculovirusDNA cotransfection meadow
6Individual Sf9 cell is cultivated in the perfect medium that contains 10% foetal calf serum in the 60mm culture dish, 27 ℃ of 50min.20 μ l lipofectin and 67.5 μ l sterilized waters are mixed, add 7.5 μ l 10ng/ μ l BaculoGold again
TMThe pAcUW51-IL12 of Linearized Baculovirus DNA and 5 μ l 100ng/ μ l.Room temperature incubation 15min.Discard substratum in the culture dish, the complete culture solution that does not contain foetal calf serum with 1.5ml replaces the nutrient solution in the culture dish.In culture dish, add lipofectin-DNA mixture, 27 ℃ of incubation 4.5h.Move and abandon above-mentioned transfection damping fluid, change to new serum free medium, cultivate 96h, gather in the crops viral supernatant for 27 ℃.
7. embodiment expresses the purifying of hIL-12 recombinant baculovirus AcNPV-hIL12
To dilute with the complete culture solution that does not contain foetal calf serum from the viral supernatant that 6. embodiment gathers in the crops, by 10
3, 10
4, 10
5, 10
6, 10
7With 10
8Extent of dilution infect 60mm culture dish (2 * 10
6Individual cell), hatch to move behind the 1.5h for 27 ℃ and abandon above-mentioned substratum, add 4ml 0.5% agarose (X-Gal that contains 0.25mg/ml), treat that it solidifies after, hatch 5d in 27 ℃, observe plaque and form.Select colourless plaque, it is taken in respectively in the perfect medium of 1ml serum-free.
With pure plaque of aseptic straw picking, agar block is moved in the EP pipe of 100 μ l serum-free mediums, inoculation one contains 2.5 * 10
6Sf9 cell and 5ml contain the 25cm of 10% foetal calf serum complete culture solution
2In the culturing bottle, 27 ℃ of incubations 5 days are infected up to cell.Determine that virus titer is 7 * 10
7Pfu/ml stores liquid in frozen pipe with this seed culture of viruses of 1ml ,-80 ℃ of preservations.This recombinant baculovirus called after AcNPV-hIL-12, and deliver the Chinese typical culture collection center preservation of Wuhan University September 14 calendar year 2001, preserving number is CCTCC-V200108.
8. embodiment expresses the evaluation of hIL-12 recombinant baculovirus AcNPV-hIL12
According to " fine works molecular biology experiment guide " (F Ao Sibai etc., 1998, Science Press, 670-671) described in method extract the recombinant baculovirus DNA that 7. embodiment is gathered in the crops.With every bottle 1.8 * 10
7The density inoculation Sf9 cell of cell is in 150cm
2In the culturing bottle.27 ℃ of incubation 2h, the infection multiplicity with 0.1 infects with recombinant baculovirus Ac-hIL12, collects supernatant after 4-5 days, and 4 ℃ 100, the centrifugal 30min of 000g obtains the virus precipitation.With extraction buffer (100mmol/L Tris, pH7.5,90mmol/LEDTA, 200mmol/L KCl) suspends, add 45 μ g/ml Proteinase Ks, 50 ℃ of insulation 2h add the 1%N-sarcosyl, 50 ℃ of incubation 2h are with isopyknic 25: 24: 1 part: chloroform: the extracting of isoamyl DNA2 time.Deposit D NA is with 1 * TE damping fluid (10mmol/L Tris Cl, 1mM EDTA, pH7.4) resuspended DNA.Add 1/10 volume 3.0mol/L sodium acetate and 2 times of volume 100% ethanol deposit D NA again.With the resuspended DNA of 1 * TE damping fluid, be stored in 4 ℃.
With recombinant baculovirus DNA is template, adopts P1, P2 and P3, two pairs of primers of P4 respectively, PCR method amplification P35 and P40 subunit cDNA fragment.PCR reaction cumulative volume 50 μ l, reaction conditions is: 94 ℃ of pre-sex change 5min, the PCR loop parameter: 94 ℃ 60 seconds, 58 ℃ 90 seconds, 72 ℃ 120 seconds, circulate 30 times, circulate in 72 ℃ of extension 7min last 1 time.Obtain 0.67kb and 1.0kb dna fragmentation respectively.The gel electrophoresis result conforms to expection, sees Figure 12.
Claims (2)
1, a kind of human interleukin-12 recombinant insect virus strain, autographa california nuclear polyhedrosis virus AcNPV-hIL12 strain is characterized in that recombinating, CCTCC NO.V200108, this recombinant virus has inserted Ro 24-7472/000-12 expression cassette, expression cassette comprises the double-promoter sequence, the P35 subunit dna encoding sequence of Ro 24-7472/000-12 and P40 subunit dna encoding sequence, the P35 subunit gene is positioned at the Polyhederin downstream, and the P40 subunit gene is positioned at P10 Promoter downstream.
2, a kind of preparation method who realizes the described human interleukin-12 recombinant insect virus strain of claim 1, its step is as follows:
A, RT-PCR amplification 667bp P35 subunit fragments and 1000bp P40 subunit fragments are cloned into pCR respectively with this two fragment
2.1 in the carrier, connect, transform, screen, identify, obtain to contain the bacterial strain of recombinant plasmid pCR35: E.coli DH5 α (pCR35), the bacterial strain that contains recombinant plasmid pCR40 and E.coli DH5 α (pCR40) by enzyme;
B, by cutting the pCR40 plasmid DNA with BglII and two kinds of enzymes of Bam HI, obtain 1000bp P40 subunit fragments, this fragment cloning in the pAcUW51 plasmid DNA of cutting through the BglII enzyme, is connected, transforms, screens, identifies the E.coli DH5 α (pAcUW51-P40) that obtains to contain recombinant plasmid pAcUW51-P40 through enzyme;
C, handle the pCR35 plasmid DNA by cutting with Eag I enzyme with T4 DNA polymerase Klenow fragment tack, obtain 667bp P35 subunit fragments, this fragment cloning to cutting through Bam HI enzyme in the pAcUW51-P40 plasmid DNA of handling with T4DNA polymerase Klenow fragment tack, is connected, transforms, screens, identifies the E.coli DH5 α (pAcUW51-IL12) that obtains to contain recombinant plasmid pAcUW51-IL12 through enzyme;
The greedy noctuid Sf9 cell in the linear autographa california nuclear polyhedrosis virus gene DNA of D, pAcUW51-IL12 plasmid DNA and deadly defective type cotransfection meadow obtains recombinant virus AcNPV-hIL12 through screening, evaluation.
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| CN103409465A (en) * | 2013-08-29 | 2013-11-27 | 武汉大学 | Preparation method and application of recombinant human leucocyte interleukin 27 |
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