CN103409465A - Preparation method and application of recombinant human leucocyte interleukin 27 - Google Patents
Preparation method and application of recombinant human leucocyte interleukin 27 Download PDFInfo
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- CN103409465A CN103409465A CN2013103825016A CN201310382501A CN103409465A CN 103409465 A CN103409465 A CN 103409465A CN 2013103825016 A CN2013103825016 A CN 2013103825016A CN 201310382501 A CN201310382501 A CN 201310382501A CN 103409465 A CN103409465 A CN 103409465A
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Abstract
The invention discloses a preparation method and application of recombinant human leucocyte interleukin 27. The preparation method comprises the following steps: amplifying EBI3 segment and P28 segment of human leucocyte interleukin 27, and cloning onto a vector pFASTbac-dual; recombining the obtained vector transformation Escherichia coli DH10Bac and a Bacmid plasmid into a recombinant transposition plasmid rBacmid, transfecting rBacmid into sf9 insect cell under the mediation of liposome to obtain recombinant baculovirus; infecting the recombinant baculovirus into the sf9 insect cell; and carrying out sf9 insect cell culture, and purifying the supernatant through a nickel affinity chromatography column and a gel chromatography column to obtain the recombinant human leucocyte interleukin 27. The human leucocyte interleukin 27 has the function of resisting hepatitis B virus infection, and can be used for preparing medicines for resisting hepatitis B virus infection. The recombinant human leucocyte interleukin 27 obtained by the method is more approximate to a natural state, and has high bioactivity; and the method has the advantages of low production cost and high yield.
Description
Technical field
The present invention relates to molecular biology, virusology and medical technical field, be specifically related to a kind of preparation method and application of recombinated interleukin-2 7.
Background technology
Interleukin-is a large type cytokines, after they are antigen presenting cell (APC) and φt cell receptor (TCR) combination, by the T emiocytosis generation activated and as immune-regulating factor, in the adjusting of propagation, growth, immunity and the inflammatory reaction of immunocyte, play an important role, prevention of disease is controlled and treatment has important clinical value.
IL-27 (IL-27) is the newcomer of IL-12 family, is found and identifies in 2002.It is a heterodimer, by the molecule 3(EBI3 of Epstein-Barr virus mediation) and two subunits of p28 form.IL-27 has some similar Biological characteristics to IL-12, and they all have the expression of higher level on antigen presenting cell (APC).When body is subject to the immunostimulation of pathogenic micro-organism and body such as TLRs and IFNs, can produce these cytokines.1998, EBI3 acceptor WSX1 was found and clones, and it is the I cytokines acceptor of NK cell and T cell expressing, and research subsequently finds that gp130 is also the acceptor of IL-27, jointly forms receptor complex with WSX1.
The existing method for preparing recombinated interleukin-2 7 is that the gene of two subunit is coupled together and is cloned on expression vector and expresses again with one section joint sequence.Recombinated interleukin-2 7 albumen of expressing like this, two subunits are not state of nature, and space structure is affected, and biological activity must be influenced.
Study and think at present, IL-27 mainly contains following functions: (1) promotes inflammatory reaction: IL-27 energy specific effect in original CD4
+The T cell, increase original CD4
+The generation of T cell IFN-γ; IL-27 can also work in coordination with the generation that IL-12 induces IFN-γ; Promote the propagation of naive T cell, and inoperative to memory t cell, the activation in TCR/CD3 is trusted in this proliferative response, and can be strengthened by CD28 and IL-12.IL-27 by with the IL-27 receptors bind after, activate STAT1, then promote the expression of Transcription Factor T-bet, IL-12R β 2 and IFN-γ etc.(2) inflammation-inhibiting reaction: Villarino etc. study discovery, WSX1 lacks mouse in the acute phase of toxoplasma gondii infection and can produce normal CE4+ and CD8+ lymphocyte and INF-gamma reaction, this reaction is enough to control parasitic breeding, yet these mouse can not be lowered the acquired immune response, and then develop into the inflammation that the CD4+ lymphocyte of lethality relies on.This pathologic reaction is to promote the lymphocytic propagation of T, increases the generation of INF-γ and IL-2, continues the CD4+ and the CD8+ lymphocyte that highly activate.(3) suppress growth and metastasis of tumours: tumor vaccine therapy mouse Colon 26 colorectal carcinomas of scholar with expression IL-27 are arranged, also find that survival time of mice extends, tumor growth is subject to obvious inhibition, induce and produce IFN-γ enhancing, more CD4+ and CD8+ cellular infiltration are arranged around tumor tissues, prompting IL-27 has the enhancing anti-tumour effect, so IL-27 has potential clinical value.
Hepatitis B virus (HBV) is a member that in hepadnavirus (Hepadnavirus) section, mammalian virus belongs to.HBV has the partially double stranded genome of a 3.2kb, comprises the reading frame of 4 styles of opening, is respectively the C gene of coding nucleocapsid, the P gene of coding polysaccharase, the S gene of coding outer membrane protein and the X protein of adjusting virogene transcriptional level.Hepatitis B virus (HBV) infects and can cause acute and Chronic Liver pathology.The liver cirrhosis that causes due to hepatitis B virus infection is died from the annual whole world or the number of liver cancer surpasses 1 million people.Reached 5 ten thousand people by the number of this virus infection every year.In worldwide, nearly 2,000,000,000 people are by the hepatitis B virus infection mistake, and it is long-term carrier of disease of hepatitis B virus that 300,000,000 5 thousand ten thousand people are arranged.Chinese 100,000,000 2 thousand ten thousand people are arranged is that (Liu Chongbai etc. (1997) Chinese public health is learned, 13:515) for viral long-term carriers.The treatment of virus disease is world's difficult problem, there is no good medicine or method.The treatment of the antiviral history of existing nearly 30 years, after the cell entry body, in host cell, copy and breed, therefore, the medicine that can suppress viral proliferation has infringement in various degree to host cell or body, makes the slower development of antiviral drug.Up to the present, there is no the antiviral drug that makes us being satisfied with fully.As the anti-hbv drug interferon-alpha, be the choice drug of current treatment chronic hepatitis B of generally acknowledging, but its curative effect only can make the case of 25～40 % obtain effective response; Lamivudine is affirmed to the restraining effect of hepatitis B virus DNA and the short term efficacy for the treatment of HBV infection, but prolonged application can induce HBV to produce resistance.
Summary of the invention
Primary and foremost purpose of the present invention is to provide a kind of preparation method of recombinated interleukin-2 7.
Another object of the present invention is to provide the application of recombinated interleukin-2 7 in preparing anti-hepatitis B virus infective medicament.
Purpose of the present invention is achieved through the following technical solutions:
A kind of preparation method of recombinated interleukin-2 7, comprise the steps:
(1) human cloning IL-27 gene: two gene fragments of the human interleukin-12 7 that increases respectively (EBI3 fragment and P28 fragment), it is cloned into to carrier pFASTbac-dual above, obtains the double expression(DE) transfer vector pFASTbac-dual-IL27 of IL-27.
(2) express the acquisition of the recombinant baculovirus of recombinated interleukin-2 7: above-mentioned transfer vector pFASTbac-dual-IL27 is transformed to intestinal bacteria DH10Bac, pFASTbac-dual-IL27 and the restructuring of Bacmid plasmid recombination to construct wherein swivel base plasmid rBacmid, liposome-mediated lower to rBacmid transfection sf9 insect cell acquisition recombinant baculovirus.
(3) at sf9 expressed in insect cells recombinated interleukin-2 7: by recombinate shape virus infection sf9 insect cell, recombinated interleukin-2 7 is expressed in the sf9 insect cell.
(4) purifying of recombinated interleukin-2 7: the sf9 insect cell of above-mentioned reorganized baculovirus infection is cultivated and centrifugally after 3 days removed cell and cell debris is collected supernatant, successively obtain recombinated interleukin-2 7 through nickel affinity chromatography column and gel chromatography column purification.
The described human cloning IL-27 of step (1) gene is preferably: take and be template from the total mRNA extracted the human blood leukocyte, first by the total cDNA of reverse transcription RCR amplification, take this cDNA is template again, with special primer, amplifies two gene fragments (EBI3 fragment and P28 fragment) of complete IL-27; Wherein, EBI3 fragment the primer P1(forward primer): 5 '-GGAATTCATGAGGAAAGGGCCCCCAGC-3 ', P2(reverse primer): 5 '-CAAGCTTTTAGTGGTG
GTGGTGGTGGTGGGAACCCTTGCCCAGGCTCATTGTGG-3 ', introduce EcoR I and Hind III restriction enzyme site, and it is cloned under the PH promotor of carrier pFASTbac-dual; P28 fragment the primer P3(forward primer): 5 '-CCTCGAGATGTTCCCAAGGCCCCCAG-3 ', P4(reverse primer): 5 '-GGCATGCTTAGTGGT
GGTGGTGGTGGTGGGAACCGGGCTGGGGGCTCAATGT-3 ', introduce Xho I and Sph I restriction enzyme site, and it is cloned under the P10 promotor of carrier pFASTbac-dual, obtains the double expression(DE) transfer vector pFASTbac-dual-IL27 of IL-27.
The purifying of the described recombinated interleukin-2 7 of step (4) is preferably: the supernatant ultrafiltration and concentration that will collect is to about 50mL(45～55mL), then go up the nickel affinity chromatography column, after 0.3M NaCl, 20mM imidazoles, 20mM pH 7.4 Tris damping fluid rinsings, utilize 0.3M NaCl, 200mM imidazoles, 20mM pH 7.4 Tris buffer solution elution, the elutriant ultrafiltration and concentration of collecting the sample peak is to about 2mL(1.8～2.2mL); Upper gel chromatography column HiLoad26/60superdex200pg, then use 20 mM Tris pH 7.4,200 mM NaCl wash-outs, collects the elutriant at sample peak, after ultrafiltration and concentration, namely obtains recombinated interleukin-2 7, cryopreservation after packing.
By recombinated interleukin-2 7 researchs that the present invention is prepared, find that it can suppress the expression of hepatitis B virus HBsAg and HBeAg, and can suppress copying of hepatitis B virus.These results show that human interleukin-12 7 has the effect of anti-hepatitis B virus infective, can be used for preparing anti-hepatitis B virus infective medicament.
A kind of anti-hepatitis B virus infective medicament, comprise human interleukin-12 7.
The present invention has following advantage and effect with respect to prior art:
The existing method for preparing recombinated interleukin-2 7 is that the gene of two subunit is coupled together and is cloned on expression vector and expresses again with one section joint sequence.Recombinated interleukin-2 7 albumen of expressing like this, two subunits are not state of nature, and space structure is affected, and biological activity must be influenced.The present invention expresses two subunits respectively, and the folding of polypeptide chain can not be affected, and the recombinated interleukin-2 7 obtained is more near native state, and biological activity is higher.And it is expensive lower than the output of mammalian cell expression system to utilize baculovirus expression system to express its output of recombinant protein.
The present invention has also found the effect of human interleukin-12 7 anti-hepatitis B virus infectives, can be used for preparing anti-hepatitis B virus infective medicament, for the treatment hepatitis B provides new direction.
Embodiment
Below in conjunction with embodiment, the present invention is done to further detailed description, but embodiments of the present invention are not limited to this.The experimental technique of unreceipted actual conditions in embodiment, usually according to the normal experiment condition, condition described in the molecular cloning third edition (New York:Cold Spring Harbor Laboratory Press, 2002), or the condition of advising according to manufacturer.
The preparation of embodiment 1 recombinated interleukin-2 7
(1) human cloning IL-27 gene: take and be template from the total mRNA extracted the human blood leukocyte, first by the total cDNA of reverse transcription RCR amplification, take this cDNA is template again, with special primer, amplifies two gene fragments (EBI3 fragment and P28 fragment) of complete IL-27; Wherein, EBI3 fragment the primer P1(forward primer): 5 '-GGAATTCATG
AGGAAAGGGCCCCCAGC-3 ', the P2(reverse primer): 5 '-CAAGCTTTTAGTGGTGGTGGTGGTG
GTGGGAACCCTTGCCCAGGCTCATTGTGG-3 ', introduce EcoR I and Hind III restriction enzyme site, and it is cloned under the PH promotor of carrier pFASTbac-dual; P28 fragment the primer P3(forward primer): 5 '-CCTCGAGATGTTCCCAAGGCCCCCAG-3 ', P4(reverse primer): 5 '-GGCATGCTTAGTGGT
GGTGGTGGTGGTGGGAACCGGGCTGGGGGCTCAATGT-3 ', introduce Xho I and Sph I restriction enzyme site, and it is cloned under the P10 promotor of carrier pFASTbac-dual, obtains the double expression(DE) transfer vector pFASTbac-dual-IL27 of IL-27.Wherein, the condition of pcr amplification: 94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 30 seconds, 59 ℃ of annealing 30 seconds, 68 ℃ were extended 1 minute, and from the sex change to the extension, carried out 30 circulations; Finally extended again 10 minutes.
(2) express the acquisition of the recombinant baculovirus of recombinated interleukin-2 7: above-mentioned transfer vector pFASTbac-dual-IL27 is transformed to intestinal bacteria DH10Bac, pFASTbac-dual-IL27 and the restructuring of Bacmid plasmid recombination to construct wherein swivel base plasmid rBacmid, liposome-mediated lower to rBacmid transfection sf9 insect cell acquisition recombinant baculovirus.
(3) at sf9 expressed in insect cells recombinated interleukin-2 7: the baculovirus infection sf9 insect cell of restructuring is made to a large amount of amplifications of virus, and recombinated interleukin-2 7 is expressed in the sf9 insect cell.
(4) purifying of recombinated interleukin-2 7: the sf9 insect cell of above-mentioned reorganized baculovirus infection is cultivated and centrifugally after 3 days to be removed cell and cell debris is collected supernatant, ultrafiltration and concentration is to about 50mL(45～55mL), then go up the nickel affinity chromatography column, after 0.3M NaCl, 20mM imidazoles, 20mM pH 7.4 Tris damping fluid rinsings, utilize 0.3M NaCl, 200mM imidazoles, 20mM pH 7.4 Tris buffer solution elution, the elutriant ultrafiltration and concentration of collecting the sample peak is to about 2mL(1.8～2.2mL); Upper gel chromatography column HiLoad26/60superdex200pg, then use 20 mM Tris pH 7.4,200 mM NaCl wash-outs, collects the elutriant at sample peak, after ultrafiltration and concentration, namely obtains recombinated interleukin-2 7, and low temperature after packing (20 ℃) is preserved.The last output of recombinated interleukin-2 7 is 6.37 grams per liter culturing cells as calculated.
(5) evaluation of recombinated interleukin-2 7: get recombinated interleukin-2 7 samples obtained above and carry out in right amount polyacrylamide gel electrophoresis, electrophoresis forwards on nitrocellulose filter after finishing.After transfer, by above-mentioned film with the TBST(20mL 1M Tris-HCl pH8.0 that contains 5% skim-milk, 8g NaCl, 0.1mL Tween20) solution soaking slowly shakes sealing, room temperature sealing 2h or 4 ℃ of sealings are spent the night.Add P28 antibody and EBI3 antibody (is purchased from R& D, the U.S.) be primary antibodie incubated at room 1 hour, with lavation buffer solution TBST, wash 3 times each shaking table jog 5～10 minutes.Add again two anti-IgG(of horseradish peroxidase to be purchased from Santa Cruz, the U.S.) diluent, shaking table jog 1h.TBST washes 5 times, each shaking table jog 5～10min.With the colour developing of horseradish peroxidase substrate, after exposure, two close specific bands of size of appearance and EBI3 and p28, illustrate that the recombinant protein obtained is human interleukin-12 7.
Embodiment 2 recombinated interleukin-2s 7 suppress hepatitis B virus HBsAg and HBeAg expresses
The HepG2.2.15 cell recombinated interleukin-2 7 of cultivation is joined to the HepG2.2.15 cell, and 24 orifice plate 0.1 μ g/ holes, put 37 ℃, 5% CO
2Incubator cultivate after 24 hours the expression level with hepatitis B virus surface antigen diagnostic kit and hepatitis B virus e antigen diagnostic kit (being purchased from Shanghai Kehua Bio-engineering Co., Ltd) detection HBsAg and HBeAg.Result shows: HBsAg level decline 65.7%, HBeAg level descends 74.1%.
Embodiment 3 rhIL-2s 7 suppress hepatitis B replication
RhIL-2 7 is joined to the HepG2.2.15 cell, and 24 orifice plate 0.1 μ g/ holes, put 37 ℃, 5% CO
2Incubator cultivate after 24 hours with the hepatitis B virogene group DNA that hepatitis B virus (HBV) nucleic acid amplification (PCR) fluorescence quantitative detection kit (detection kit is purchased from Shenzhen base biotechnology limited-liability company) detects and nucleocapsid is connected, primer P5:5 '-ATCCTGCTGCTATGCCTCATCTT-3 ' and P6:5 '-ACAGTGGGGAAAGCCCT
ACGAA-3 ', probe: 5 '-TGGCTAGTTTACTAGTGCCATTTTG-3 ', reaction conditions: 50 ℃ of circulations in 2 minutes; 95 ℃ of circulations in 10 minutes; 95 ℃ 15 seconds, 60 ℃ were carried out 40 circulations in 60 seconds.Result shows that the level of cccDNA descends 55.2%.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
SEQUENCE LISTING
<110 > Wuhan University
<120 > a kind of preparation method of recombinated interleukin-2 7 and application
<130> 1
<160> 7
<170> PatentIn version 3.5
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ggaattcatg aggaaagggc ccccagc 27
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<223 > primer P2
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caagctttta gtggtggtgg tggtggtggg aacccttgcc caggctcatt gtgg 54
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<213> Artificial Sequence
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<223 > primer P3
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cctcgagatg ttcccaaggc ccccag 26
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<223 > primer P4
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ggcatgctta gtggtggtgg tggtggtggg aaccgggctg ggggctcaat gt 52
<210> 5
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<223 > primer P5
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atcctgctgc tatgcctcat ctt 23
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<223 > primer P6
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acagtgggga aagccctacg aa 22
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tggctagttt actagtgcca ttttg 25
Claims (5)
1. the preparation method of a recombinated interleukin-2 7, is characterized in that comprising the steps:
(1) human cloning IL-27 gene: the EBI3 fragment of the human interleukin-12 7 that increases respectively and P28 fragment, it is cloned into to carrier pFASTbac-dual above, obtain the double expression(DE) transfer vector pFASTbac-dual-IL27 of IL-27;
(2) express the acquisition of the recombinant baculovirus of recombinated interleukin-2 7: above-mentioned transfer vector pFASTbac-dual-IL27 is transformed to intestinal bacteria DH10Bac, pFASTbac-dual-IL27 and the restructuring of Bacmid plasmid recombination to construct wherein swivel base plasmid rBacmid, liposome-mediated lower to rBacmid transfection sf9 insect cell acquisition recombinant baculovirus;
(3) at sf9 expressed in insect cells recombinated interleukin-2 7: by recombinate shape virus infection sf9 insect cell, recombinated interleukin-2 7 is expressed in the sf9 insect cell;
(4) purifying of recombinated interleukin-2 7: the sf9 insect cell of above-mentioned reorganized baculovirus infection is cultivated and centrifugally after 3 days removed cell and cell debris is collected supernatant, successively obtain recombinated interleukin-2 7 through nickel affinity chromatography column and gel chromatography column purification.
2. the preparation method of recombinated interleukin-2 7 according to claim 1, it is characterized in that: the described human cloning IL-27 of step (1) gene is: take and be template from the total mRNA extracted the human blood leukocyte, first by the total cDNA of reverse transcription RCR amplification, take this cDNA is template again, with special primer, amplifies EBI3 fragment and the P28 fragment of complete IL-27; Wherein, EBI3 fragment the primer P1:5 '-GGAATTCATGAGGAAAGGGCCCCCAGC-3 ', P2:5 '-CAAGCTTTTAGTGGTGGTGGTGGTGGTGGGAACCCTTGCCCAGGCTCATTGTGG-
3 ', introduce EcoR I and Hind III restriction enzyme site, it is cloned under the PH promotor of carrier pFASTbac-dual; P28 fragment the primer P3:5 '-CCTCGAGATGTTCCCAAGGCCCCCAG-3 ', P4:5 '-GGCATGCTTAGT
GGTGGTGGTGGTGGTGGGAACCGGGCTGGGGGCTCAATGT-3 ', introduce Xho I and Sph I restriction enzyme site, and it is cloned under the P10 promotor of carrier pFASTbac-dual, obtains the double expression(DE) transfer vector pFASTbac-dual-IL27 of IL-27.
3. the preparation method of recombinated interleukin-2 7 according to claim 1, it is characterized in that: the purifying of the described recombinated interleukin-2 7 of step (4) is: the supernatant ultrafiltration and concentration to 45 that will collect～55mL, then go up the nickel affinity chromatography column, after 0.3M NaCl, 20mM imidazoles, 20mM pH 7.4 Tris damping fluid rinsings, utilize 0.3M NaCl, 200mM imidazoles, 20mM pH 7.4 Tris buffer solution elution, the elutriant ultrafiltration and concentration at collection sample peak is 1.8～2.2mL extremely approximately; Upper gel chromatography column HiLoad26/60superdex200pg, then use 20 mM Tris pH 7.4,200 mM NaCl wash-outs, collects the elutriant at sample peak, after ultrafiltration and concentration, namely obtains recombinated interleukin-2 7, cryopreservation after packing.
4. the application of human interleukin-12 7 in preparing anti-hepatitis B virus infective medicament.
5. an anti-hepatitis B virus infective medicament, is characterized in that: comprise human interleukin-12 7.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106474458A (en) * | 2017-01-05 | 2017-03-08 | 重庆医科大学 | Application in treatment C. difficile infection for the interleukin-22 7 |
| CN114736878A (en) * | 2021-01-07 | 2022-07-12 | 秦皇岛摩登狗生物科技有限公司 | Recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 and application thereof |
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| CN1357622A (en) * | 2001-11-02 | 2002-07-10 | 武汉大学 | Human interleukin-12 recombinant insect virus strain and its prepn |
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| CN1357622A (en) * | 2001-11-02 | 2002-07-10 | 武汉大学 | Human interleukin-12 recombinant insect virus strain and its prepn |
Non-Patent Citations (2)
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| 曹艳华 等: ""由白细胞介素-29介导的白细胞介素-27抑制乙型肝炎病毒复制机制的研究"", 《2012年鄂粤微生物学学术年会——湖北省暨武汉微生物学会成立六十年庆祝大会论文集 , 2012 年》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106474458A (en) * | 2017-01-05 | 2017-03-08 | 重庆医科大学 | Application in treatment C. difficile infection for the interleukin-22 7 |
| CN114736878A (en) * | 2021-01-07 | 2022-07-12 | 秦皇岛摩登狗生物科技有限公司 | Recombinant baculovirus co-expressing porcine interferon L3 and porcine interleukin 22 and application thereof |
| CN114736878B (en) * | 2021-01-07 | 2024-04-19 | 秦皇岛摩登狗生物科技有限公司 | Co-expression pig interferon L3 and pig interleukin 22 recombinant baculovirus and application thereof |
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Application publication date: 20131127 |