CN102993292A - AFP (Alpha Fetal Protein) recombinant protein and in-intro recombinant expression method - Google Patents
AFP (Alpha Fetal Protein) recombinant protein and in-intro recombinant expression method Download PDFInfo
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Abstract
The invention relates to an AFP (Alpha Fetal Protein) recombinant protein and an in-intro recombinant expression method, and in particular relates to an artificial signal peptide-AFP-609aa-6his recombinant protein and an in-intro recombinant expression method of a mammal transient expression system. The protein sequence of the recombinant protein is shown in SEQ ID NO: 1. The in-intro recombinant expression method comprises the following steps of: cloning a recombinant protein gene to a pcDNA 3.1+ carrier through double enzyme digestion to establish pcDNA3.1+-AFP expression plasmids, and carrying out plasmid extraction; b, transfecting the pcDNA3.1+-AFP plasmid obtained by using extraction into CHO-S suspension cells by PEI (polyethyleneimine) and carrying out recombinant protein expression; and c, separating and purifying for 7 days to obtain the recombinant protein. The artificial synthesized AFP protein prepared by using the large-scale transient suspension CHO-S expression system has the activity similar to that of the natural AFP protein and is higher in purity, low in endotoxin content, free of pollution from animal-based protein and capable of being expected to be used as a tumor antigen vaccine for liver cancer immunotherapy.
Description
[technical field]
The present invention relates to the method for AFP recombinant protein and in-vitro recombination expression, be specifically related to recombinant protein and the Mammals transient expression system in-vitro recombination expression method of a kind of manual signal peptide-AFP-609aa-6his.
[background technology]
Primary hepatocellular carcinoma (primary hepatocellular carcinoma, PHC are called for short liver cancer) is one of common malignant tumour, and a whole world year morbidity case is approximately 1,200,000 people.At present, the general curative effect of liver cancer treatment is still very undesirable, and the methods for the treatment of of liver cancer mainly contains surgical excision, liver transplantation etc. clinically.The patient's of surgical excision recurrence rate is very high, and 3 years recurrence rates of postoperative are that 50%, 5 year recurrence rate is 70%.Liver Transplantation for Treatment although especially Living Donor Liver Transplantation makes some progress, is faced with the problems such as donor source shortage.Therefore, clinically in the urgent need to the method for the new Hepatoma therapy of research and development.In recent years, along with the fast development of immunology and molecular biology mechanism theoretical investigation, and people are to the intensification of liver cancer cell knowledge of biological, and the liver cancer immunity treatment becomes new tumor therapeuticing method.The at present known target antigen that may become the liver cancer immunity treatment has: AFP, MAGE family, HBV/HCV virus antigen, and some oncogenes (myc, fos, ras) etc.
AFP is a kind of specific proteins that occurs in human fetus's phase blood.It is synthetic in the liver cell of fetus.AFP content in the HAS is atomic, but at the hepatocyte function abnormal, when suffering from primary hepatocellular carcinoma, can occur again AFP in the serum and raise especially, so clinically usually by the inspection of the AFP auxiliary diagnosis as primary hepatocellular carcinoma.50%～80% liver cancer patient is expressed AFP, and the concentration in serum may surpass 1 mg/ml.In human development's process, specific CTL clone for AFP in the T cell bank does not remove, felicity condition and method can be with it activation, Butterfield etc. are wrapped in 4 AFP epitope peptides in the incomplete freund adjuvant, patient HCC positive to 6 routine HLA A2.1, that express AFP carries out intradermal immunization, and 5 routine patients have shown AFP peptide specific T cell amplification to 3 epitope peptides wherein.Fundamental research shows that DC is the strongest full-time antigen presenting cell of function in the body, but because the interior DC great majority of liver cancer patient body are in immature state, and there is obstacle in its function, induces the ability of Allogenic Lymphocytes propagation to reduce.Ripe by external evoked DC, and the epitope peptide in load AFP source, the ability of activation specific CTL killing hepatoma cell.
Mammalian expression system has unique advantage aspect the start signal of albumen, processing, secretion, the glycosylation, is fit to the high molecular weight protein of the expressed.Modify the exogenous protein that produces by reprocessing after the mammalian cell translation, outclass the eukaryotic expression systems such as prokaryotic expression system and yeast, insect cell aspect active, closer to natural protein.Because biotech company had released preferably expression system accordingly to the research and development of mammalian expression system in recent years, had greatly improved the expression amount of foreign protein in the lactation system.Along with increasingly matureization of suspension system, the success of now extensive instant expression of exogenous protein can reach mg to g rank at present.Chinese hamster ovary cell CHO is as expressive host, its advantage have following some: genetic background is clear, and physiological metabolism is stable; Approach with people's sibship, foreign protein is modified accurately; Transgenosis and vector expression system are perfect; The tolerance shearing force is convenient to large scale culturing; Confirmed as safe genetically engineered recipient cell by U.S. FDA.
[summary of the invention]
In order to prepare on a large scale highly purified AFP albumen, the invention provides the recombinant protein of a kind of manual signal peptide-AFP-609aa-6his and the method for preparation thereof.
For achieving the above object, design the recombinant protein of a kind of manual signal peptide-AFP-609aa-6his, it is characterized in that its protein sequence is shown in SEQ ID NO:1.
The base sequence of manual signal peptide is shown in SEQ ID NO:2.
The present invention also comprises a kind of method with the described recombinant protein of Mammals transient expression system vivoexpression, comprises the synthetic of recombinant protein gene,
It is characterized in that the method includes the steps of:
A. utilize gene synthesis technology, synthetic manual signal peptide-AFP-609aa-6his sequence
B. manual signal peptide-AFP-609aa-6his gene order is cloned in the pcDNA3.1+ carrier by double digestion, makes up the pcDNA3.1+-AFP expression plasmid, and carry out plasmid extraction,
C. the pcDNA3.1+-AFP plasmid that extracting is obtained advances the expression that the CHO-S suspension cell carries out recombinant protein with the PEI transfection,
D.7 the recombinant protein that separation and purification obtains behind the sky.
Come separation and purification with the Ni-post.
Carry out described double digestion with Hind III and EcoR I.
Above-mentioned recombinant protein can be applied to cancer treatment drug.
The present invention mainly comprises the content of four aspects: (1) utilizes gene synthesis technology, synthetic recombinant AFP dna sequence dna, then with sequence clone in the pcDNA3.1+ carrier, make up the pcDNA3.1+-AFP plasmid, prepare on a large scale the pcDNA3.1+-AFP plasmid; (2) use CHO suspension expression system to express recombinant AFP albumen; (3) use Ni-NTA post separation and purification recombinant AFP albumen from the suspension cell supernatant liquor.(4) AFP albumen behind the purifying is stimulated dendritic cell (dendritic cells, DC),, form CTL and kill and wound the HepG2 cell t cell activation with the DC cell after the activation, detect kill rate with mtt assay.
The object of the invention is close with natural A FP albumen by the synthetic AFP protein-active of extensive instantaneous suspension CHO-S expression system preparation, and the purity that obtains is higher, endotoxin content is low, and the pollution of non-animal derived property albumen can be expected to the tumour antigen vaccine as the liver cancer immunity treatment.
[description of drawings]
Fig. 1 is the plasmid pcDNA3.1+ collection of illustrative plates of mammalian expression system,
Fig. 2 is that the enzyme of pcDNA3.1+-AFP is cut evaluation agarose electrophoresis figure,
Fig. 3 is recombinant AFP albumen figure behind the SDS-PAGE analysis purifying,
Fig. 4 is recombinant AFP albumen figure behind the Western-blot analysis purifying,
Fig. 5 is that mtt assay detects the HepG2 Growth of Cells data plot of processing through CTL.
The specifying information of the collection of illustrative plates among Fig. 1 is:
CMV promotor (CMV promoter): bases 232-819
T7 promotor/primer binding site (T7 promoter/priming site): bases 863-882
Multiple clone site (Mutiple cloning site): bases 895-1010
The primer binding site of anti-chain (pcDNA3.1/BGH reverse priming site): bases 1022-1039
BGH polyadenylic acid sequence (BGH polyadenylation sequence): bases 1028-1252
F1 replication origin (f1 origin): bases 1298-1726
Sv40 early promoter and replication origin (SV40 early promoter and origin): bases 1731-2074
Neomycin resistance gene (opening code-reading frame) Neomycin resistance gene (ORF): bases 2136-2930
SV40 polyadenylic acid signal (SV40 polyadenylation signal): bases 3104-3234
PUC replication origin (pUC origin): bases 3617-4287 (complementary strand complementary strand)
Ampicillin resistant gene Ampicillin resistance gene (bla): bases 4432-5428(complementary strand complementary strand)
Opening code-reading frame (ORF): bases 4432-5292 (complementary strand complementary strand)
Ribosome bind site (Ribosome binding site): bases 5300-5304 (complementary strand complementary strand)
Bla promotor Bla promoter (P3): bases 5327-5333 (complementary strand complementary strand)
[embodiment]
In order to make purpose of the present invention, technical scheme and advantage clearer, the present invention is further elaborated.Production unit among the application all is the common equipment of this area, should be appreciated that specific embodiment described herein only in order to explain the present invention, is not intended to limit the present invention.
Embodiment 1:
1. research material: recombinant plasmid pUC57-AFP provides for outsourcing service company; DNA marker, restriction enzyme, T4 ligase enzyme etc. all are purchased from Takara; PcDNA3.1+, CHO-S and Ni-NTA resin are Invitrogen company product; CHO-S cell culture medium FreeStyle CHO is the product of Gibco.PEI is the Polysciences product.
2. workflow:
(1) by Hind III and EcoR I double digestion pUC57-AFP, the AFP that downcuts is inserted into same pcDNA3.1+ by Hind III and EcoR I double digestion by the T4 ligase enzyme, be built into expression vector pcDNA3.1+-AFP.
(2) preparation competence DH5 α transforms pcDNA3.1+-AFP, is tiled in LB Amp+ culture plate, and 37 ℃ of incubators spend the night.Choose 3 mono-clonals, put in the 5ml LB nutrient solution, 37 ℃ are shaken soon and spend the night.The little 5ml of taking out culture and cut evaluation with extract product enzyme is in a small amount chosen the culture of positive colony.Choose a positive colony and carry out the 500ml amplification cultivation of spending the night, then bacterium liquid is poured in the aseptic centrifuge tube, 4 ℃ centrifugal, centrifugal 15 minutes of 6000g.Take out greatly according to taking out greatly test kit Standard Operating Procedure process plasmid, it is aseptic to obtain at last 2.5mg, high superhelix, low endotoxin, the high-quality plasmid of A260/280=1.82.Hind III and EcoR I, 1% agarose gel electrophoresis is identified.The result shows that fragment is accurate.Electrophoresis result has the external source fragment at 2000 bp places, has shown that recombinant transfer plasmid successfully constructs, as shown in Figure 2.
(3) 24h before the transfection, inoculation 1L density is 0.8x10
6/ ml suspension cell CHO-S.Transfection same day, cell counting guarantees that cell density is 2x10
6/ ml can carry out transfection.Prepare dna-PEI mixture after room temperature leaves standstill 10min, joins mixture in the cell, puts into 37 ℃, 5%CO
2Incubator is cultivated.Behind the 24h, add TN1.Cultivate after six days, take out cell suspension.
(4) cell suspension carries out centrifugally, filters.Utilize the Ni-NTA column separating purification to obtain corresponding protein supernatant liquor, and by SDS-PAGE and Western blot analytical pure purity of protein.The result has band and purity to reach more than 95% at the 70kD place, has shown the success of recombinant AFP protein expression as shown in Figure 3 and Figure 4.
(5) use blood cell separator and gather health donors peripheral blood mononuclear cell 30ml.Adopt the method for Ficoll density gradient separation to separate mononuclearcell (PBMC).Use the physiological saline washed cell, the PBMC after the washing is suspended in 1640 serum free mediums, places six orifice plates, every hole 2 ml, and cell concn is 2x10
6/ ml.37 ℃, 5%CO
2Incubator is cultivated 2h, keeps attached cell, collects non-adherent cell, and is as lymphocyte, frozen.Every hole adds IL-4, RPMI-1640 nutrient solution 2 m1 of the rhGM-CSF, the 50ng/ml that contain 1000u/m1,37 ℃, 5%CO
2Incubator is cultivated.Added on the 5th day that the rAFP of 0,50,100,150 ug/ml stimulates DC behind the purifying, adding TNF-a after 24 hours stimulates the DC chemotactic ripe.The 7th day collecting cell is then with continuous normal saline washing three times.
(6) collect the DC cell as irritation cell.Recover frozen non-adherent cell as reacting cells.With reacting cells and irritation cell in the combined inoculation of 20:l ratio in 24 orifice plates, every hole l m1,37 ℃, 5%CO
2Cultivate 5 d, the cell of collection is the effector cell.Collect the HepG2 cell of logarithmic phase as target cell, with effector cell and target cell in the combined inoculation of 20:1 ratio in 96 orifice plates, final volume 200ul.Establish simultaneously single target cell group, the single effect cell is established 3 multiple holes for every group.37 ℃, 5%CO
2After cultivating 24 h, add MTT (final concentration 0.5 mg/m1), 37 ℃, 5%CO
2Cultivate 4h, centrifugal 5 min of 1000 rpm/min inhale and abandon the 200ul supernatant, fill into 150ul DMSO, and 10 min that vibrate fully after the blue precipitation of dissolving, detect the A490 value with enzyme-linked immunosorbent assay instrument at 490 nm places.According to absorbance, calculate cell survival rate.Cell survival rate=[experimental group A average-background group A average]/[blank group A average-background group A average] * 100 %.Its result as shown in Figure 5.
Claims (6)
1. the recombinant protein of a manual signal peptide-AFP-609aa-6his is characterized in that its protein sequence is shown in SEQ ID NO:1.
2. recombinant protein as claimed in claim 1 is characterized in that the base sequence of manual signal peptide is shown in SEQ ID NO:2.
3. the method with the described recombinant protein of Mammals transient expression system vivoexpression claim 1 comprises the synthetic of recombinant protein gene,
It is characterized in that the method includes the steps of:
A. the recombinant protein gene is cloned in the pcDNA3.1+ carrier by double digestion, makes up the pcDNA3.1+-AFP expression plasmid, and carry out plasmid extraction,
B. the pcDNA3.1+-AFP plasmid that extracting is obtained advances the expression that the CHO-S suspension cell carries out recombinant protein with the PEI transfection,
C.7 the recombinant protein that separation and purification obtains behind the sky.
4. the method with the described recombinant protein of Mammals transient expression system vivoexpression claim 1 as claimed in claim 3 is characterized in that coming separation and purification with the Ni-NTA post.
5. the method with the described recombinant protein of Mammals transient expression system vivoexpression claim 1 as claimed in claim 3 is characterized in that carrying out described double digestion with Hind III and EcoR I.
6. a recombinant protein claimed in claim 1 is used for the application of cancer treatment drug.
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| CN2012105456651A CN102993292A (en) | 2012-12-14 | 2012-12-14 | AFP (Alpha Fetal Protein) recombinant protein and in-intro recombinant expression method |
| CN201310683033.6A CN103755811B (en) | 2012-12-14 | 2013-12-13 | A kind of AFP recombiant protein and external high efficiency recombinant expressed method |
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| CN116515000A (en) * | 2023-06-30 | 2023-08-01 | 迦进生物医药(上海)有限公司 | HFE fusion proteins and uses thereof |
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| CN107987147A (en) * | 2016-10-26 | 2018-05-04 | 王惠琴 | A kind of MG53 polymers preparation method and its usage |
| CN107881156A (en) * | 2017-11-21 | 2018-04-06 | 上海海洋大学 | Express GCRV spike protein VP56 recombinant baculovirus and application |
| CN113667685B (en) * | 2018-08-07 | 2023-02-28 | 康码（上海）生物科技有限公司 | Signal peptide related sequence and application thereof in protein synthesis |
| CN114262718B (en) * | 2021-12-28 | 2022-11-15 | 武汉爱博泰克生物科技有限公司 | Cell secretion expression method and application of human Ly6d recombinant protein |
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| CN1826350A (en) * | 2003-07-22 | 2006-08-30 | 梅里麦克药品公司 | Non-glycosylated human alpha-fetoprotein, methods of production, and uses thereof |
| CN1993469A (en) * | 2004-07-14 | 2007-07-04 | 生物系统股份有限公司 | Recombinant alpha-fetoprotein, method and means for preparation thereof, compositions on the base thereof and use thereof |
| CN102219856A (en) * | 2011-05-18 | 2011-10-19 | 哈尔滨医科大学 | Vascular Endothelial Growth Factor (VEGF) acceptor 2/CD3 bispecific single-chain antibody |
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Patent Citations (4)
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| CN1826350A (en) * | 2003-07-22 | 2006-08-30 | 梅里麦克药品公司 | Non-glycosylated human alpha-fetoprotein, methods of production, and uses thereof |
| CN1993469A (en) * | 2004-07-14 | 2007-07-04 | 生物系统股份有限公司 | Recombinant alpha-fetoprotein, method and means for preparation thereof, compositions on the base thereof and use thereof |
| CN102686730A (en) * | 2009-11-16 | 2012-09-19 | 亚赛特生物技术私人有限公司 | Recombinant antibody vector |
| CN102219856A (en) * | 2011-05-18 | 2011-10-19 | 哈尔滨医科大学 | Vascular Endothelial Growth Factor (VEGF) acceptor 2/CD3 bispecific single-chain antibody |
Non-Patent Citations (2)
| Title |
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| 匡志鹏等: "pcDNA3.1-mAFP真核表达载体的构建及其在293细胞中的表达", 《中国现代医学杂志》, vol. 17, no. 9, 30 May 2007 (2007-05-30) * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116515000A (en) * | 2023-06-30 | 2023-08-01 | 迦进生物医药(上海)有限公司 | HFE fusion proteins and uses thereof |
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| CN103755811A (en) | 2014-04-30 |
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