CN107602705B - A kind of fusion protein and its application being used to detect cell PD1 expressions based on interactions between protein - Google Patents

Abstract

The present invention relates to a kind of fusion protein and its applications being used to detect cell PD1 expressions based on interactions between protein, belong to technical field of biological.Fusion protein of the present invention, it is characterised in that by PDL1 albumen combined area segment 18-132 amino acids and GFP green fluorescent proteins by connect peptide in conjunction with by obtain.Experiment shows that fusion protein of the invention both ensure that the combination of PDL1 and PD1, also ensure that will not because of in conjunction with and initiator is dead.The fusion protein for PD1 detect, compared with traditional antibody test, have the advantages that it is efficient, at low cost and sensitiveer, be it is a kind of quickly, efficiently, accurately detection PD1 new method.

Description

It is a kind of based on interactions between protein be used for detect cell PD1 expressions fusion protein and It is applied

Technical field

The present invention relates to biotechnology, a kind of fusion protein more particularly to detection cell PD1 expressions and It is applied.

Background technology

Programmed death receptor -1 (programmed cell death protein-1, PD1/CD279) is important and exempts from Epidemic disease checkpoint inhibits T thin by the effect with two ligand PDL1 (B7-H1/CD274) and PDL2 (B7-DC/CD273) The activation of born of the same parents and the generation of cell factor play vital effect on the peripheral tolerance for maintaining body.

Immunologic test point is originally the molecule to shield in human immune system, plays similar brake, prevents T thin Inflammation damnification etc. caused by born of the same parents' excessive activation.And tumour cell is exempted from using this characteristic of human immune system by overexpression Epidemic disease checkpoint molecule inhibits human immune system's reaction, human immunity monitoring and killing is escaped, to promote the life of tumour cell It is long.

Clinically research and most widely used immunologic test point inhibitor include CTLA-4, PD1 and PDL1 monoclonal antibody at present, By inhibiting immunologic test point activity, the immune brake in release tumor microenvironment to reactivate T cell and answer the immune of tumour Effect is answered, to reach antineoplastic action.

On lymphocyte the expression of PD1 be clinical examination, medication guide important symbol object (biomarker) it One, the antibody of presently commercially available detection PD1 has the disadvantage that：1. of high cost, the R＆D costs and production cost of antibody are higher；2. Insensitive, since PD1 antibody drugs develop, the antibody of efficient and sensible is chiefly used in developing curative drug；3. non-specific binding, Antibody itself is also easy to produce non-specific binding, influences result.4. testing result be difficult to unification, due to each antibody cog region not Together, binding force is different, it is thus possible to directly result in the different generations of result there is a phenomenon where different due to antibody.Therefore, it is badly in need of Develop the cheap streaming antibody surrogate product of stability and high efficiency.

Since PDL1 and PD1 are naturally occurring ligand and receptor, and most can really be reacted with PDL1 detections PD1 can Can be by the lymphocyte that immunologic test point is influenced the case where, but after being combined with PD1 due to PDL1, it can be dead with startup program It dies, therefore simply still infeasible in the method for PDL1 as detection PD1.

Invention content

For the defects of above-mentioned field, a kind of fusion protein is provided, which both ensure that the combination of PDL1 and PD1, Also ensure that will not because of in conjunction with and initiator is dead.

Application the present invention also provides the fusion protein in detecting PD1 expressions simultaneously.With traditional antibody test Compare, have the advantages that it is efficient, at low cost and sensitiveer, be it is a kind of quickly, efficiently, accurately detect PD1 new method.

A kind of fusion protein, it is characterised in that green by the combined area segment 18-132 amino acids and GFP of PDL1 albumen Color fluorescin by connect peptide in conjunction with by obtain.

The connection peptide sequence is：GGGGSGGGGSGGGGS, HHHHPGGSVKKR, SAPGTP, SAPGTPSR or PG.

The amino acid sequence of the fusion protein such as SEQ ID NO：Shown in 1.

PDL1-GFP:AFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLK VQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAGGGGSGGGGSGGGGSMSKGE ELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGVQCFARYPDHMKQHD FFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKN GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDEL YK

Application of the above-mentioned fusion protein in detecting cell PD1 expressions.

The present invention is based on the three-dimensional structures of PD1 and PDL1 interactions, analyze the amino acid region for participating in combining on PDL1 It is 18 to 132 amino acids sequences, the combined area segment of PDL1 albumen is melted with GFP green fluorescent proteins by connecting peptide Close, experiment shows that fusion protein of the invention both ensure that the combination of PDL1 and PD1, also ensure that will not because of in conjunction with and cause Programmed death.

Compared with traditional antibody test, have the advantages that it is efficient, at low cost and sensitiveer, be it is a kind of quickly, efficiently, Accurately detect the new method of PD1.

Description of the drawings

The three-dimensional structure of Fig. 1 PDL1 and PD1 interactions,

Fig. 2 electrophoretic analysis PCR amplification as a result,

Wherein 1：PDL1^18-132；2：PD1 overall lengths；3：GFP；4：PET28a,

Fig. 3 SDS-PAGE electrophoretic analysis protein expressions as a result,

Fig. 4 Mass Spectrometric Identifications as a result,

Fig. 5 SDS-PAGE electrophoretic analysis protein expressions and purification result,

Wherein 1:Empty carrier precipitates；2:PDL1-GFP albumen precipitations；3:Empty carrier supernatant；4:PDL1-GFP albumen supernatants； 5:Flow through sample；6:Washing buffer elution samples；7:The preceding 2mL of Elution I elutions；8:Elution I elute sample Product；9:Elution II elution samples

Fig. 6 SDS-PAGE electrophoretic analysis protein expressions and purification result,

1:Empty carrier supernatant；2:PDL1-GFP albumen supernatants；3:Flow through sample；4、5:Washing buffer elute sample Product；6:The preceding 2mL of Elution II elutions；7、8:Elution II elution samples；9:H₂O elution samples,

Fig. 7 SDS-PAGE electrophoretic analysis protein expressions and purification result,

1:Empty carrier supernatant；2:PDL1-GFP albumen supernatants；3:Flow through sample；4、5:Washing buffer elute sample Product；6:The preceding 2mL of Elution II elutions；7、8:Elution II elution samples；9:H₂O elution samples,

Fig. 8 PDL1-GFP protein purifications (G25 desalinations),

Fig. 9 SDS-PAGE electrophoretic analysis protein expressions and purification result,

Wherein 1-5:Eluting peak；6:Albumen before purification

Figure 10 flow cytometer detection PBMC apoptosis situations,

Figure 11 flow cytomery PD1 expressions,

Wherein 1- negative controls, 2- streaming antibody, 3-PDL1-GFP,

Figure 12 fluorescence microscope cell PD1 expressions.

Specific implementation mode

With reference to embodiment, the present invention is described in further detail.

Following experiment materials are commercially available.

Embodiment 1

Experimental method：

1, the regional structure combined analysis is participated on PDL1

Three-dimensional structure (Fig. 1) when according to PDL1 the and PD1 interactions of parsing analyzes the amino acid area for participating in combining on PDL1 Domain is 18 to 132 amino acids sequences (in frame), wherein red mark is binding site.Participate in the amino acid sequence combined For¹⁸AFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NA¹³², it is labeled as：PDL1^18-132。

PDL1-GFP fusion protein amino acid sequences design

PDL1-GFP:AFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEED LKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA

GGGGSGGGGSGGGGS(HHHHPGGSVKKR)(SAPGTP)(SAPGTPSR)(PG)*

MSKGEELFTG VVPILVELDG DVNGHKFSVS GEGEGDATYG KLTLKFICTT GKLPVPWPTL VTTFGYGVQC

FARYPDHMKQ HDFFKSAMPE GYVQERTIFF KDDGNYKTRAEVKFEGDTLV NRIELKGIDF KEDGNILGHK

LEYNYNSHNV YIMADKQKNG IKVNFKIRHN IEDGSVQLAD HYQQNTPIGD GPVLLPDNHY LSTQSALSKD

PNEKRDHMVL LEFVTAAGIT HGMDELYK

*：Sequence in () is the alternative sequence of lower setting-out partial sequence, to connect peptide

2, PDL1 is built^18-132With the fusion protein of GFP

It is reacted using PCR, using cDNA sequence as masterplate, expands PDL1^18-132And GFP sequences

PCR system is as follows：

PCR conditions are as follows：

3, using methods of homologous recombination, by the PDL1 of amplification arrived^18-132It is linked, is marked with GFP segments and carrier For：PDL1-GFP, reaction system are as follows：

50 DEG C are incubated 15min, cooled on ice 1min, and 2 μ L recombinant products is taken to be transferred in DH5 α competent cells, and 950 μ are added 37 DEG C of 200rpm of L LB culture mediums cultivate 1h, and 100 μ L is taken to be coated on the tablet of the LB containing 100 μ g/mL kanamycins, 37 DEG C of cultures Overnight.

4, positive colony is transferred in Rosetta (DE3) competent cell

Correct positive colony is sequenced, conservation simultaneously extracts plasmid, is transferred in Rosetta (DE3) competent cell.

5, protein induced expression

1) bacterium is connect in 5mL LB liquid mediums (K resistances), it is 37 DEG C, after 220rpm cultivates 12h, 5mL culture solutions is whole It is transferred in 300mL LB liquid mediums (K resistances), cultivates about 3h, OD600 reaches 0.5, and it can be 0.1- that IPTG final concentrations, which are added, 1.0mM, temperature can be 16 DEG C -30 DEG C, overnight incubation 12h；

2) 10000rpm centrifuges 10min and collects thalline, and precipitation is dissolved in the buffer solution of 20mM Tris-HCl pH 8.2, into Row ultrasonication (ice bath), power 65%, time 5min, super 3s stop 5s.10000rpm centrifuges 20min after ultrasound, collects supernatant, Precipitation is resuspended with a small amount of 20mM Tris-HCl pH 8.2；

3) SDS-PAGE analyzes protein expression situation.

6, protein purification

1) after Ni columns rinse 5 column volumes with 20mM Tris-HCl, loading, and collect and flow through sample；

2) it is washed away with the Washing Buffer of 5 column volumes (20mM Tris-HCl, 100mM imidazoles, 500mM NaCl) Foreign protein collects sample；

3) it is washed with the Eluton Buffer I (20mM Tris-HCl, 250mM imidazoles, 500mM NaCl) of 5 column volumes Destination protein collects sample；

4) it is washed with the Eluton Buffer II (20mM Tris-HCl, 500mM imidazoles, 500mM NaCl) of 5 column volumes Destination protein collects sample；

5) SDS-PAGE analyzes the albumen situation in each collection sample.

7, it is detected the case where PBMC apoptosis in incubation

1) PBMC processing：1000rpm centrifuges 5min, collects cell, is resuspended in and is washed once, then with OKM+ with 0.5mL PBS In 5%FBS, PDL1-GFP albumen, final concentration of 10 μ g/mL, 37 DEG C of 5%CO is added₂Culture is for 24 hours；

2) PBMC is carefully drawn, 2000rpm centrifuges 10min；

3) it is washed once with 500 μ L PBS, 2000rpm centrifuges 10min；

4) cell is resuspended with 100 μ L PBS again, is separately added into 7AAD and Annexin V and carries out single dye, experimental group adds simultaneously Enter 7AAD and Annexin V and carry out double dyes, is protected from light and is incubated 30min；

5) 1mL PBS, 2000rpm is added and centrifuges 10min；

6) it is washed once with 1mL PBS again, 2000rpm centrifuges 10min, is finally resuspended in spare in 500 μ L PBS.

Experimental result：

1.PCR expands PDL1^18-132And GFP segments

Using the cDNA of the RNA reverse transcriptions of H358 as template, PDL1 is amplified respectively^18-132Segment and GFP segments, (Fig. 2) again It is linked, is transferred in DH5 α competent cells through homologous recombination with pET28a, after sequencing is correct, then be transferred to Rosetta (DE3) In, it is expressed；

2.PDL1-GFP protein extractions

The bacterial strain of Rosetta (DE3) is transferred to as negative control using pET28a, and protein expression situation is as shown in figure 3, and carrier It compares, the supernatant precipitation of PDL1-GFP albumen has an apparent expression band (Fig. 3 arrows are signified), and Mass Spectrometric Identification result is such as Shown in Fig. 4, the albumen of expression is PDL1 segments.

3.PDL1-GFP protein purification process optimizations

1) Ni column purifications

Expand cultivating system, largely to carry out subsequent experimental after preparation PDL1-GFP protein purifications；Protein purification result is such as Shown in Fig. 5 (swimming lane 8 and 9), position shown in arrow is PDL1-GFP albumen can obtain PDL1-GFP albumen in eluent Thick sterling, but concentration is relatively low；After being concentrated with super filter tube, shown in albumen such as Fig. 6 (swimming lane 7 and 8), although, destination protein The concentration of PDL1-GFP increases, but other miscellaneous bands also display, therefore, it is necessary to improve purification condition, to obtain high quality Thick sterling.

2) anion exchange column purification

The PDL1-GFP of prokaryotic expression is further purified in anion-exchange column, the results are shown in Figure 7 (swimming lane 7- 9), compared with Fig. 6, purer PDL1-GFP albumen can be obtained.

3) G25 desalting columns carry out desalting processing to ion exchange to PDL1-GFP albumen

Gained sample in (2) is further purified in G25 desalting columns, (swimming lane 1,2 and as a result as shown in Figure 8 and Figure 9 4), compared with Fig. 9 before purification (swimming lane 6), purer PDL1-GFP albumen can be obtained.

4.PDL1-GFP albumen will not cause the apoptosis of PBMC in a short time

PD1/PDL1 --- programmed death receptor/programmed death ligand is gained the name because of its function, micro- in tumour immunity In environment, the PDL1 on tumour cell by PD1 on bind lymphocytes, and then influences lymphocyte and functions, therefore, The most important condition that PD1 is detected with PDL1 is cannot to cause apoptosis, leads to Apoptosis.External apoptosis tests table Bright (Figure 10)：There is no the PBMC of any processing to compare, the cell proportion of early apoptosis and late apoptic is 2.2%；PDL1- is added When after GFP albumen for 24 hours, the cell proportion of early apoptosis and late apoptic is 2.1%, similar to control group；After 48h, PBMC is early The cell proportion of phase apoptosis and late apoptic is 2.2%, is illustrated, PDL1-GFP albumen in a short time, can not cause PBMC's Apoptosis can be used for the detection of cell PD1.

5.PDL1-GFP Protein Detection PD1 expressions --- flow cytometer

Respectively with commercially available PDL1 flow cytometer detections antibody (Thermo Scientific, PD-1Monoclonal Antibody, MA5-16134 the PDL1-GFP) and herein built is detected the PD1 expressions on PBMC, as a result as shown in figure 11,1 It is commercially available PDL1 flow cytometer detections antibody for negative control, 2, can detects that the presence of PD1, positive ratio are 67.9%, 3 are The PD1+ cells that PDL1-GFP is detected, ratio are 99.6% far above the ratio that streaming physical examination measures；The comparison of two methods It is shown in Table 1, PD1 is detected with PDL1-GFP, has many advantages, such as at low cost, efficient and high sensitivity, therefore be vitro detection PD1 The best approach.

1 distinct methods of table detect the comparison of PD1

6.PDL1-GFP Protein Detection PD1 expressions --- fluorescence microscope

The PD1 expressions on PBMC are detected with PDL1-GFP albumen, the result of fluorescence microscopy microscopic observation is such as Shown in Figure 12, green PBMC is the cell for expressing PD1.

In conclusion compared with antibody detection method, flow cytometer detection and fluorescence microscope can be passed through with PDL1-GFP The expression of PD1 is analyzed in detection, and has the advantages that at low cost, speed soon and high sensitivity, therefore, with PDL1- It is effective new method that GFP, which detects PD1,.

Sequence table

<110>Beijing sources Ding Chengtai Bioisystech Co., Ltd

<120>A kind of fusion protein and its application being used to detect cell PD1 expressions based on interactions between protein

<130>PP17072-DCT

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 368

<212> PRT

<213> programmed cell death protein-1

<400> 1

Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly

1 5 10 15

Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp

20 25 30

Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile

35 40 45

Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr

50 55 60

Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala

65 70 75 80

Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg

85 90 95

Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys

100 105 110

Val Asn Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

115 120 125

Gly Ser Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile

130 135 140

Leu Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser

145 150 155 160

Gly Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe

165 170 175

Ile Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr

180 185 190

Thr Phe Gly Tyr Gly Val Gln Cys Phe Ala Arg Tyr Pro Asp His Met

195 200 205

Lys Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln

210 215 220

Glu Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala

225 230 235 240

Glu Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys

245 250 255

Gly Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu

260 265 270

Tyr Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys

275 280 285

Asn Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly

290 295 300

Ser Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp

305 310 315 320

Gly Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala

325 330 335

Leu Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu

340 345 350

Phe Val Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys

355 360 365

Claims (3)

1. a kind of fusion protein, it is characterised in that by 18-132 amino acids combined area segment and the GFP greens of PDL1 albumen Fluorescin by connect peptide in conjunction with by obtain.

2. fusion protein according to claim 1, the connection peptide sequence are：GGGGSGGGGSGGGGS、 HHHHPGGSVKKR, SAPGTP, SAPGTPSR or PG.

3. fusion protein according to claim 2, the amino acid sequence such as SEQ ID NO of the fusion protein：Shown in 1.