CN1544637A - Method for preparing mB7.1-GPI fusion proteins and their uses - Google Patents
Method for preparing mB7.1-GPI fusion proteins and their uses Download PDFInfo
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Abstract
The invention provides the preparation and application of mB7.1-GPI fusion protein, an eukaryotic expression system (pcDNA3.1(+)-mB7.1-GPI) of B7.1(mB7.1-GPI), preparing mB7.1-GPI fusion proteins. They can anchor on tumor cell membrane, and the experiment proves that the anchoring has considerably stability; keeps bioactivity of immune protein, and has the functions of stimulating multiplication of mice splenic cells and secreting IL-2 and IFN-Y genes. The mB7.1-GPIs anchor the tumor cells to make tumor vaccine able to inhibit tumor growth of tumor-bearing mice and prolong the living time of the tumor-bearing mice, and its mechanism has relation with the activity of animal immune cells NK and CTL in tumor vaccine enhancement test and the cell genes IL-2 and IFN-gama secreted.
Description
Affiliated field
The invention belongs to biotechnology, relate generally to the preparation of glycosylation phosphatidylinositols grappling type mouse B7.1 fusion rotein and the preparation and the application aspect oncotherapy of this fusion protein film finishing knurl seedling.
Background technology
Antitumor knurl seedling is the main policies of tumor immune gene therapy, and to carrying out finishing to strengthen its immunogenicity and immunizing potency from body or allogeneic tumor cell, the knurl seedling is modified and mainly realized by the gene transfection means at present in knurl seedling preparation process.As Hodge etc.
[1]With expressing in the B7.1 gene importing tumour cell, costimulating factor is provided, can strengthen the anti tumor immune response of knurl seedling; Shrayer etc.
[2]With SEA gene transfection mouse B16 melanoma cell, make it to express SEA, use this oncocyte and make the knurl seedling, shown stronger antitumor action.
Though the gene transfection method plays very vital role on tumor immune gene therapy, the gene transfection method also has its limitation
[3], be difficult for transfection as primary cell, transfection efficiency is low, integrate the unstable of the degraded of back gene and expression thereof and prepare time-consuming and clinical application difficult.Therefore be necessary to seek and be suitable for primary cell transfection, transfection efficiency height, easy, the clinical infection protocol of easily applying of preparation.
By genetic engineering means with glycosylation phosphatidylinositols (GPI) and target protein matter chimeric expression, Zhi Bei target protein mass-energy enough anchors on the various kinds of cell film thus, does not thisly make method that target protein expresses on target cell membrane albumen infection protocol (protein transfer) by the gene transfection means
[4]
The albumen infection protocol has many advantages
[4]: (1) does not rely on cell self proliferation potential and transfection, so can be used for the transfection of multiple primary cell, has nothing to do with the type of cell and the source of histoorgan; (2) also there are certain difficulty in the cotransfection of complex gene and coordinate expression in same cell, and the albumen transfection can allow multiple protein simultaneously or in a sequence to be anchored on cell surface, are suitable for the preparation of multiple immune molecule film finishing knurl seedling; (3) external, the immune molecule that can be used for the albumen transfection by a large amount of preparations of gene engineering method, after these immune molecules and patient's autologous tumor cell are hatched jointly, can prepare knurl seedling more easily through the autologous tumor cell of immune molecule finishing, need not in autologous tumor cell, to do individual gene transfection, therefore bigger clinical application potentiality are arranged.
This research prepares mB7.1-GPI by gene engineering method, is anchored to mouse T cell lymphoma cell (EL-4), prepares the knurl seedling, observes its antitumor action.The method domestic and foreign literature that this research prepares mB7.1-GPI does not all have report.
Summary of the invention
An object of the present invention is to prepare the mB7.1-GPI fusion rotein, SEQ ID NO:1 is the nucleotide sequence of mB7.1-GPI fusion gene.
Another object of the present invention is by the albumen infection protocol, and the mB7.1-GPI fusion rotein is anchored on the oncocyte film, and the preparation surface of cell membrane is modified the knurl seedling.
The present invention is anchored to mouse T cell lymphoma cell (EL-4) with the mB7.1-GPI fusion rotein, preparation knurl seedling.
The method that the present invention prepares the mB7.1-GPI fusion rotein mainly may further comprise the steps:
(1) make up hPLAP-1 the 11st exon cloning vector pGEM-hPLAP-1 11exon (it comprises GPI grappling signal sequence), as template, amplification GPI grappling signal sequence.
(2) NSX-mB7-1 is a template, amplification mB7-1 gene extracellular region sequence.
(3) utilize the SOEing reaction, two sequences that increase are coupled together.
(4) make up carrier for expression of eukaryon pcDNA3.1 (+)/mB7.1-GPI of this junction fragment, order-checking is correct.
(5) use the liposome transfection method, with pcDNA3.1 (+)/mB7.1-GPI transfection in Chinese hamster ovary celI, with G418 screening G418 resistance clone, with flow cytometry, screening positive clone screens a high-expression clone, and its positive rate is 96.6%, fluorescence intensity is 34.8, names this clone to be CHO/pcDNA3.1 (+)-mB7.1-GPI.
(6) with immunoaffinity chromatography method purifying expressing protein, analyze the about 67KDa of demonstration purified product molecular weight through SDS-PAGE and Western Blot.
Another purpose of the present invention is the application of knurl seedling aspect oncotherapy of making,
MB7.1-GPI fusion rotein provided by the invention has film grappling function, mB7.1 can be anchored on the tumor cell membrane altogether, and have suitable stability.
Characteristics of the present invention are: mB7.1-GPI can be anchored on the tumor cell membrane, experimental results show that this grappling has suitable stability; The biologic activity that has kept immune protein simultaneously is in the external function that stimulation mouse boosting cell propagation is arranged and secrete IL-2, the IFN-γ factor.MB
7-1-GPI grappling tumour cell, the knurl seedling of making, can suppress the tumor growth of tumor-bearing mice and prolong survival time of tumor-bearing mice, its mechanism and knurl seedling strengthen laboratory animal immunocyte NK and CTL activity and immune stimulating activity emiocytosis IL-2, the IFN-gamma cells is factor-related.
Description of drawings
The agarose gel electrophoresis of Fig. 1 hPLAP-1 gene the 11st exon PCR product
The recombinate gel electrophoresis of pGEM-hPLAP-1 11exon plasmid DNA and double digestion product thereof of Fig. 2
The sequencing result of Fig. 3 pGEM-hPLAP-1 11exon
1% agarose gel electrophoresis of Fig. 4 PCR product
The gel electrophoresis that Fig. 5 pGEM-mB7.1-GPI enzyme is cut product
Fig. 6 pcDNA3.1 (+)/mB7.1-GPI enzyme is cut the gel electrophoresis of product
The sequencing result of Fig. 7 pcDNA3.1 (+)/mB7.1-GPI
Fig. 8 mB7.1 antigen is expressed in the Chinese hamster ovary celI with gene transfection
The immunofluorescence laser confocal microscope observations of Fig. 9 rotaring redyeing gene Chinese hamster ovary celI
Chinese hamster ovary celI surface mB7.1 expression of results before and after Figure 10 PI-PLC handles
The mCD80-CNBr-Separose 4B affinitive layer purification of Figure 11 mB7.1-GPI
Figure 12 SDS-PAGE analyzes Chinese hamster ovary celI mB7.1-GPI Expression of Fusion Protein and purification result (Coomassie blue stain)
Figure 13 Western Blot analyzes Chinese hamster ovary celI mB7.1-GPI Expression of Fusion Protein and purification result
The immunofluorescence laser confocal microscope observations of Figure 14 mB7.1-GPI grappling EL-4 cell
The tumor growth curve of Figure 15 tumor-bearing mice after accepting different preparation for treating
Active detect (the LDH method for releasing) of the different preparation for treating group of Figure 16 mouse NK
The CTL activity (LDH method for releasing) of the different preparation for treating group of Figure 17 mouse
The lifetime of Figure 18 tumor-bearing mice after accepting different preparation for treating
Embodiment
The present invention is further illustrated below in conjunction with embodiment and accompanying drawing, and non-limiting the present invention.
All related datas all adopt the SPSS software package to carry out statistical analysis in this experiment, and survival rate relatively adopts the Log-rank check, and other measurement data adopts variance analysis (F check) and relatively q check in twos.
The structure of purifying one hPLAP-1 the 11st exon cloning vector of structure, expression and the product of embodiment one mB7.1-GPI gene eukaryotic expression vector:
1. hPLAP-1 gene the 11st exon size of the electrophoretic analysis pcr amplification of amplification hPLAP-1 gene the 11st exon product is 307bp, 1.5% agarose gel electrophoresis result is referring to Fig. 1, wherein M:100bp dna molecular size indicates., the PCR product of swimming lane 1:hPLAP-111 exon.
2. the enzyme of reorganization pGEM-hPLAP-1 11exon plasmid DNA is cut and is identified reorganization pGEM-hPLAP-1 11exon plasmid DNA EcoRI and XhoI double digestion, enzyme is cut product with 1.5% agarose gel electrophoresis, the goal gene size is 307bp, the result is referring to Fig. 2, wherein M1, M2:DNA molecular size are marked, the PCR product of swimming lane 1:hPLAP-1 11 exons, the enzyme of swimming lane 2:pGEM-hPLAP-1 11 exons is cut product.
3. the sequential analysis of amplification gene
HPLAP-1 gene the 11st exon sequence
[4]See Genbank (M19159.1), the standard sequence among our sequencing result (referring to Fig. 3) and the Genbank (M19159.1) shows that relatively upstream 200bp and standard sequence are in full accord.The GPI grappling signal peptide gene sequence of hPLAP-1 C-terminal is positioned at upstream 150bp, so hPLAP-1 gene the 11st exon sequence of being cloned can be used for the structure of mB7.1-GPI fusion gene.
Two, the structure of the cloning and expression carrier of mB7.1-GPI gene
1. the electrophoretic analysis of amplification mB7.1 gene film outskirt product
With the pLNSX-mB7-1 plasmid DNA is template, with primer P3 and P4, the mB7.1 that pcr amplification goes out to be used for to be connected with hPLAP-1 gene the 11st exon GPI grappling signal peptide gene sequence fragment is because of film outskirt fragment, size is 761bp, referring to Fig. 4, wherein M1, M2:DNA molecular size sign, the PCR product of swimming lane 1:hPLAP-1 grappling signal peptide sequence, the PCR product of swimming lane 2:mB7.1 gene extracellular region sequence; The PCR product of swimming lane 3:mB7.1-GPI gene order.
2. the electrophoretic analysis of the GPI grappling signal peptide sequence of amplification coding hPLAP-1 gene
With pGEM-hPLAP-1 11exon plasmid DNA is template, amplifies the fragment of the GPI grappling signal peptide sequence of coding hPLAP-1 gene with primer P5 and P6, and size is 155bp, and electrophoresis result is referring to Fig. 4.
3.mB7.1-GPI the electrophoresis of fusion gene PCR product is identified
PCR product with above-mentioned 1,2 is a template, and with primer P3 and P6 amplification membrane grappling type costimulatory molecules mB7.1-GPI fusion gene, its size is 891bp, and electrophoresis result is referring to Fig. 4.
4.pGEM-mB7.1-GPI and the enzyme of pcDNA3.1 (+)/mB7.1-GPI is cut evaluation
Cloning vector pGEM-mB7.1-GPI and expression vector pcDNA3.1 (+)/mB7.1-GPI are behind EcoRI and XhoI double digestion, the goal gene size is 891bp, enzyme is cut 1% agarose gel electrophoresis result of product referring to Fig. 5,6, M:DNA molecular size sign among Fig. 5, the enzyme of swimming lane 1:pGEM-mB7.1-GPI plasmid is cut product; M:DNA molecular size sign among Fig. 6, swimming lane 1:pcDNA3.1 (+) plasmid, the enzyme of swimming lane 2:pcDNA 3.1 (+)/mB7.1-GPI plasmid is cut product, swimming lane 3:pcDNA3.1 (+)/mB7.1-GPI plasmid.
The sequencing result of (5.pcDNA3.1+)/mB7.1-GPI
Through with Genbank in mB7.1 gene (GeneBank X60958) sequence
[5]Compare with hPLAP-1 gene order (M19159.1), this research clone's mB7.1-GPI fusion gene comprises the gpi signal peptide-coding sequence (referring to Fig. 7) of mB7.1 gene film outskirt encoding sequence and hPLAP-1 gene.
Three, the expression and purification of mB.1-GPI fusion gene
(1) the Chinese hamster ovary celI clone's of expression mB.1-GPI fusion rotein qualification result
1.FCM behind art: pcDNA3.1 (+)/mB7.1-GPI transfection CHO cell, chosen 30 G418 resistance clones, FCM detects the expression of mB7.1.The positive expression of 13 strains, positive rate 21.9%～96.6%, the positive intensity of mean fluorecence is 4.76～34.8; Wherein the expression of 1 strain is higher than other positive expression strains, and for efficiently expressing the clone, positive rate is 96.6%, and average fluorescent strength is 34.8 (referring to Fig. 8), names this clone to be CHO/pcDNA3.1 (+)-mB7.1-GPI.No mB7.1 expresses in the Chinese hamster ovary celI of wild Chinese hamster ovary celI and empty carrier pcDNA3.1 (+) transfection, the Chinese hamster ovary celI called after CHO/pcDNA3.1 (+) of transfection empty carrier pcDNA3.1 (+).
2. the immunofluorescence laser confocal microscope is observed: CHO/pcDNA3.1 (+)-mB7.1-GPI cell climbing sheet, the laser confocal microscope of immunofluorescence is observed, see that surface of cell membrane is green fluorescence (FITC mark) (referring to Fig. 9), illustrate that the mB7.1-GPI fusion rotein is the film expression type, wild Chinese hamster ovary celI, CHO/pcDNA3.1 (+) cell are negative.
3.PI-PLC result: CHO/pcDNA3.1 (+), CHO/pcDNA3.1 (+)-mB7.1-GPI cell is handled through PI-PLC, and FCM detects PI-PLC
[6]The variation of cell surface mB7.1 before and after handling, the result shows: cell surface did not all have the expression of mB7.1 before and after CHO/pcDNA3.1 (+) cell PI-PLC handled; CHO/pcDNA3.1 (+)-mB7.1-GPI cell is after PI-PLC handles, and the mB7.1 The positive expression rate reduces to 9.46% from 96.6%, and fluorescence intensity reduces to 4.68 from 34.8, and referring to Figure 10, wherein (a) is for before handling, (b) for after handling.
(2) purifying protein qualification result (SDS-PAGE and Western Blot analyze)
1. the immunoaffinity chromatography process is referring to Figure 11: elution process has only a main peak, illustrates that the antibody chromatography column is special figure.
2.SDS-PAGE and the purified product behind Western Blot result: CHO, CHO/pcDNA3.1 (+), CHO/pcDNA3.1 (+)-mB7.1-GPI product of cell lysis and CHO/pcDNA3.1 (+)-mB7.1-GPI product of cell lysis immunoaffinity chromatography is analyzed through SDS-PAGE (Coomassie blue stain) (ginseng Figure 12) and Western Blot (ginseng Figure 13).M among ginseng Figure 12: molecular weight of albumen sign, swimming lane 1: the m7.1-GPI albumen of purifying; Swimming lane 2:CHO product of cell lysis, swimming lane 3:CHO/pcDNA3.1 (+)/mB7.1-GPI product of cell lysis, swimming lane 4:CHO/pcDNA3.1 (+) product of cell lysis.Swimming lane 1 among ginseng Figure 13: the m7.1-GPI albumen of purifying, swimming lane 2:CHO product of cell lysis, swimming lane 3:CHO/pcDNA3.1 (+)/mB7.1-GPI product of cell lysis, swimming lane 4:CHO/pcDNA3.1 (+) product of cell lysis.
The result shows: the purified product of CHO/pcDNA3.1 (+)/mB7.1-GPI product of cell lysis and immunoaffinity chromatography can be seen the band of an about 67KDa of molecular weight; And this band does not all appear in CHO, CHO/pcDNA3.1 (+) product of cell lysis; The purity of purifying protein reaches more than 95%.
The applied research of embodiment two mB7.1-GPI fusion roteins
1.mB7.1-GPI fusion rotein is to the grappling effect of tumor cell membrane
MB7.1-GPI and EL-4 oncocyte are hatched, and the laser confocal microscope of immunofluorescence is observed and seen that surface of cell membrane is green fluorescence (referring to Figure 14), illustrates that mB7.1-GPI albumen can anchor on the EL-4 oncocyte film.MB7.1-GPI and EL-4 oncocyte are hatched behind the 4hr 4 ℃ jointly and are placed 0h, 4h or 8h, and FCM detects.The fluorescence intensity of 0h, 4h, 8h sample is respectively 13.7,12.7,10.6 after the grappling, and the positive cell number percentage is respectively 95.4%, 91.3%, 85%.4h, 8h sample and 0h sample relatively, fluorescence intensity and positive cell number percentage reduce not obvious, show that fusion rotein and EL-4 tumour cell are hatched after, can be anchored on the tumor cell membrane, and this grappling has suitable stability.The 4 ℃ of EL-4/mB7.1-GPI cells of placing the 0h samples and EL-4 cell are made tumour-cell vaccine through mitomycin deactivation (final concentration is 100ug/ml, 37 ℃, 5%CO2 incubation 1hr), are used for that lymphproliferation response and secrete cytokines detect and immunity
2. tumour cell knurl seedling stimulates the effect of mouse boosting cell propagation and secrete cytokines
The multiplication capacity (PI represents with proliferation index) of EL-4/mB7.1-GPI knurl seedling stimulation mouse boosting cell and the amount of secreting leukocytes mesonium-2 (IL-2) and IFN-(IFN-γ) are than EL-4 knurl height of seedling, and difference has significance (P＜0.05).
Table 1 knurl seedling stimulates mouse boosting cell propagation and secrete cytokines ability
Group IL-2 IFN-γ PI
AEL-4 22.9±1.4 41±3.2 1.98±0.7
BEL-4/mB7.1-GPI
a 508.8±22.1 805±13.5?3.43±0.6
IL-2, IFN-γ: mean ± SD pg/ml, PI:mean ± SD,
aCompare with the A group
3. tumor vaccine is to the therapeutic action of tumor-bearing mice
3.1 tumor vaccine to the restraining effect of tumor-bearing mice tumor growth in the right hind subcutaneous injection 1 * 10 of C57BL/6 mouse
6Individual wild-type EL-4 cell.After 7-10 days, find to have palp tumour.Each organizes mouse Subcutaneous tumor upgrowth situation referring to Figure 15.The average tumor diameter of EL-4 knurl seedling treatment group mouse is organized less than PBS, but its differences does not have significance (P＞0.05); The average tumor diameter of EL-4/mB7.1-GPI knurl seedling group mouse is little than EL-4 knurl seedling treatment group and PBS group, and difference has significance (P＜0.05).
3.2 the NK activity of tumor-bearing mice splenocyte
Adopt the LDH method for releasing that the splenocyte of difference treatment group tumor-bearing mice is carried out the active detection of NK.Target cell is YAC-1, and the ratio of effector cell and target cell was respectively 25: 1,50: 1 and 100: 1.Referring to Figure 16, the result shows: EL-4/mB7.1-GPI knurl seedling group NK activity is high than EL-4 knurl seedling group and PBS group, and difference has significance (P＜0.05).
3.3. the CTL activity of tumor-bearing mice splenocyte
3.4 the lifetime of tumor-bearing mice
Except being used to detect NK and active 3 mouse of CTL,, observe its lifetime to remaining 5 mouse.The result shows: EL-4 knurl seedling group (mean lifetime 40.2 ± 1.3 days) is compared with the PBS group and is prolonged slightly lifetime, but there was no significant difference (P＞0.05); EL-4/mB
7-1-GPI knurl seedling group mouse (mean lifetime 58 ± 3.5 days) is compared with EL-4 knurl seedling group with the PBS group and prolongs lifetime, significant difference (P＜0.05) is arranged, referring to Figure 18.
The partial reference document that the present invention relates to:
1.odge?JW，Abrams?S，schlom?J，et?al.Induction?of?antitumor?immunity?byrecombinant?vaccinia?viruses?expressing?B7-1?and?B7-2?costimulatorymolecules.Cancer?Res，1994，54：5552-5555.
2.hrayer?DP，Kouttab?N，Hearing?VJ，et?al.Immunization?of?mice?withmelanoma?cells?transfected?to?secrete?the?superantigen，staphylococcalenterotoxin?A.Cancer-Immunol-Immunother.1998?Mar；46(1)：7-13
3.ykocinski?ML，Kaplan?DR，Medof?ME.Antigen-presenting?cellengineering.AJP?January，1996，148(1)：1-16.
4.illan?JL.Molecular?Cloning?and?Sequence?Analysis?of?Human?PlacentalAlkaline?Phosphatase.J.Biol.Chem.1986，261(7)：3112-3115.
5.Freeman GJ, GrayGS, GimmiCD, et al.Structure, Expression, and T CellCostimulatory Activity of the Murine Homologue of the Human BLymphocyte Activation Antigen B7.J Exp Med, 1991,174:625-6SEQ ID NO:1 mB7.1-GPI fusion gene sequence
1 atggcttgca?
attgtcagtt?
gatgcaggat?
acaccactcc?
tcaagtttcc?
agtccaagg
61 ctcattcttc?tctttgtgct?gctgattcgt?ctttcacaag?tgtcttcaga?tgttgatgaac
121?aactgtccaa?gtcagtgaaa?gataaggtat?tgctgccttg?ccgttacaac?tctcctcatg
181?aagatgagtc?tgaagaccga?atctactggc?aaaaacatga?caaagtggtg?ctgtctgtca
241?ttgctgggaa?actaaaagtg?tggcccgagt?ataagaaccg?gactttatat?gacaacacta
301?cctactctct?tatcatcctg?ggcctggtcc?tttcagaccg?gggcacatac?agctgtgtcg
361?ttcaaaagaa?ggaaagagga?acgtatgaag?ttaaacactt?ggctttagta?aagttgtcca
421?tcaaagctga?cttctctacc?cccaacataa?ctgagtctgg?aaacccatct?gcagacacta
481?aaaggattac?ctgctttgct?tccgggggtt?tcccaaagcc?tcgcttctct?tggttggaaa
541?atggaagaga?attacctggc?atcaatacga?caatttccca?ggatcctgaa?tctgaattgt
601?acaccattag?tagccaacta?gatttcaata?cgactcgcaa?ccacaccatt?aagtgtctca
661?ttaaatatgg?agatgctcac?gtgtcagagg?acttcacctg?ggaaaaaccc?ccagaagacc
721?ctcctgatag?caagaacaca?tacaccgcct?gcgacctggc?gccccccgcc?ggcaccaccg
781?acgccgcgca?cccggggcgg?tccgtggtcc?ccgcgttgct?tcctctgctg?gccgggaccc
841?tgctgctgct?ggagacggac?tgctccctga?ctagag
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use to greatest extent, and these equivalent form of values fall within the application's appended claims institute restricted portion equally.In addition, the preferred specific embodiments of front should be understood that only to illustrate, but not limits the scope of the invention by any way.
Claims (6)
1.mB7.1-GPI the preparation of fusion rotein and application thereof is characterized in that: preparation mB7.1-GPI fusion rotein, SEQ ID N0:1 is the nucleotide sequence of mB7.1-GPI fusion gene.
2. the preparation of mB7.1-GPI fusion rotein according to claim 1 and application thereof is characterized in that: by the albumen infection protocol, the mB7.1-GPI fusion rotein is anchored on the oncocyte film, the preparation surface of cell membrane is modified the knurl seedling.
3. the preparation of mB7.1-GPI fusion rotein according to claim 2 and application thereof is characterized in that: the mB7.1-GPI fusion rotein is anchored to mouse T cell lymphoma cell (EL-4), preparation knurl seedling.
4. the preparation of mB7.1-GPI fusion rotein according to claim 1 and application thereof is characterized in that: the method for preparing the mB7.1-GPI fusion rotein mainly may further comprise the steps:
(1) make up hPLAP-1 the 11st exon cloning vector pGEM-hPLAP-1 11exon (it comprises GPI grappling signal sequence), as template, amplification GPI grappling signal sequence.
(2) pLNSX-mB7-1 is a template, amplification mB7-1 gene extracellular region sequence.
(3) utilize the SOEing reaction, two sequences that increase are coupled together.
(4) make up carrier for expression of eukaryon pcDNA3.1 (+)/mB7.1-GPI of this junction fragment, order-checking is correct.
(5) use the liposome transfection method, with pcDNA3.1 (+)/mB7.1-GPI transfection in Chinese hamster ovary celI, with G418 screening G418 resistance clone, with flow cytometry, screening positive clone screens a high-expression clone, and its positive rate is 96.6%, fluorescence intensity is 34.8, names this clone to be CHO/pcDNA3.1 (+)-mB7.1-GPI.
(6) with immunoaffinity chromatography method purifying expressing protein, analyze the about 67KDa of demonstration purified product molecular weight through SDS-PAGE and Western Blot.
5. the preparation of mB7.1-GPI fusion rotein according to claim 1 and application thereof is characterized in that: mB7.1-GPI fusion rotein provided by the invention has film grappling function, mB7.1 can be anchored on the tumor cell membrane altogether, and have suitable stability.
6. the preparation of mB7.1-GPI fusion rotein according to claim 1 and application thereof is characterized in that: the application of knurl seedling aspect oncotherapy of making.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100406564C (en) * | 2006-02-28 | 2008-07-30 | 浙江大学 | Method for preparing polygene transfection tumor cell strain and its tumor vaccine |
| CN102391377A (en) * | 2011-11-01 | 2012-03-28 | 孙嘉琳 | Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein |
| CN106755028A (en) * | 2016-12-24 | 2017-05-31 | 山东明鑫集团有限公司 | A kind of method for building up of the outer grappling expression vector of cell membrane |
| WO2023088246A1 (en) * | 2021-11-17 | 2023-05-25 | 上海君赛生物科技有限公司 | Membrane surface protein containing gpi anchor region |
-
2003
- 2003-11-18 CN CNA2003101087786A patent/CN1544637A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100406564C (en) * | 2006-02-28 | 2008-07-30 | 浙江大学 | Method for preparing polygene transfection tumor cell strain and its tumor vaccine |
| CN102391377A (en) * | 2011-11-01 | 2012-03-28 | 孙嘉琳 | Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein |
| CN102391377B (en) * | 2011-11-01 | 2013-11-06 | 孙嘉琳 | Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein |
| CN106755028A (en) * | 2016-12-24 | 2017-05-31 | 山东明鑫集团有限公司 | A kind of method for building up of the outer grappling expression vector of cell membrane |
| WO2023088246A1 (en) * | 2021-11-17 | 2023-05-25 | 上海君赛生物科技有限公司 | Membrane surface protein containing gpi anchor region |
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