CN107974461A

CN107974461A - A kind of construction of eukaryotic expression vector method of high efficient expression anti-hEGFR genes

Info

Publication number: CN107974461A
Application number: CN201810048426.2A
Authority: CN
Inventors: 任桂萍; 李德山; 刘春香; 王宇阳
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2018-01-18
Filing date: 2018-01-18
Publication date: 2018-05-01

Abstract

The invention discloses a kind of construction of eukaryotic expression vector method of high efficient expression anti hEGFR genes, belong to biology field.Clone and highly effective eukaryon expression carrier structure for recombinant human epidermal growth factor acceptor antibody (anti hEGFR) gene, using the plasmid of the full-length genes of hEGFR containing anti as template, design primer monoclonal antibody light and heavy chain, IgKappa Leader and IRES EGFP are inserted on carrier for expression of eukaryon to strengthen protein expression and facilitate follow-up separation to test, the albumen anti hEGFR of eukaryotic expression are successfully obtained by aforesaid way, it will play remarkable result in terms of cancer caused by treatment EGFR is overexpressed.

Description

A kind of construction of eukaryotic expression vector method of high efficient expression anti-hEGFR genes

Technical field

The present invention relates to a kind of construction of eukaryotic expression vector method of high efficient expression anti-hEGFR genes, belongs to molecule life Thing field.

Background technology

In recent years, malignant tumour endangers human health as a kind of stubborn disease always, and the whole world there are about 1400 every year People more than ten thousand suffers from cancer, and has 8,200,000 people to die of cancer.Wherein, Chinese cancer number of suffering from is 3,070,000 people, and has about 2,200,000 People is dead.At present, the treatment of cancer is main or based on radiation and chemotherapy, but chemicotherapy is often to the damage that human body is brought Unrepairable, such as radioactivity liver, pulmonary fibrosis.EGF-R ELISA (epidermal growth factor Receptor, EGFR) as a kind of tyrosine kinase receptor, cell signal transmission can be carried out, has studied confirmation, EGFR mistakes Degree expression and some tumours, such as non-small cell lung cancer (NSCLC), colorectal cancer, breast cancer are closely related.In cancer patient In, after reducing EGFR by human intervention, the state of an illness of cancer patient can be obviously improved.The EGFR antibody of humanization can be right The EGFR ligand-binding sites point on Skin Squamous Cell Carcinoma cell (A431) surface is closed, so as to weaken or block the signal in EGFR downstreams Conduction, reaches the growth and invasion and attack for suppressing tumour, promotes the purpose of Apoptosis.This just makes anti-hEGFR in treatment EGFR mistakes There is very big application value in terms of cancer caused by expression, one is provided for the treatment of cancer using EGFR as drug target A new thinking.

This research clones anti-hEGFR's using anti-EGFR humanized antibodies gene as template by molecule clone technology Total length heavy chain and full-length light chains, are connected to peedual carrier for expression of eukaryon, to obtain eukaryotic expression by the gene cloned Recombinant human epidermal growth factor acceptor antibody is used for the treatment of cancer.

The content of the invention

The present invention provides a kind of construction of eukaryotic expression vector method of high efficient expression anti-hEGFR genes, eucaryon is obtained Protein expression vector Peedual-IRES-EGFP-hEGFR plasmids, structure is as described in Figure 11.

The purpose of the present invention can be achieved through the following technical solutions：

The clone of 1.hEGFR heavy chain of antibody and light chain gene.

2. by hEGFR antibody light chains and heavy chain PCR products glue reclaim after purification with pMD18-T simple cloning vectors Connection.

3. convert recombinant cloning vector and extract light chain and heavy chain recombinant clone plasmid pK-LC and pK-HC respectively.

It is prepared by the digestion of 4.LC, HC, IRES EGFP gene fragment and pKlight, pee12.4, pee6.4 carrier.

The structure of 5.Pee12.4-LC, pee6.4-HC-IRES EGFP recombinant vectors.

The structure of 6.Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon.

7. go extracting and the concentration mensuration of endotoxic peedual-IRES-EGFP-hEGFR plasmids.

Brief description of the drawings

Fig. 1 is the constructing plan schematic diagram of peedual-IRES-EGFP-hEGFR recombinant vectors.

Fig. 2 is hEGFR heavy chains and light chain PCR result figures.

Fig. 3 is the PCR purified products of LC, HC fragment.

Fig. 4 is digestion and the PCR qualification result figures of LC-T, HC-T recombinant plasmid.

Fig. 5 is the digestion preparation production of LC, HC, IRES EGFP gene fragment and pKlight, pee12.4, pee6.4 carrier Thing recycles result.

Fig. 6 is the digestion identification and PCR identifications of PK-LC, PK-HC recombinant plasmid.

Fig. 7 is that the double digestion product of each recombinant plasmid recycles result.

Fig. 8 is pee12.4-LC, pee6.4-HC-IRES EGFP digestions and PCR are identified.

Fig. 9 identifies for peedual-IRES-EGFP-hEGFR digestions and PCR.

The expression SDS-PAGE that Figure 10 is anti-hEGFR is analyzed

Figure 11 eukaryotic protein expression vector Peedual-IRES-EGFP-hEGFR plasmid construct figures

Embodiment

Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, is conventional method unless otherwise specified.Test material used in following embodiments, is certainly unless otherwise specified What routine biochemistry reagent shop was commercially available.Escherichia coli E.coil DH5 α, CHO-K1 cell line, carrier for expression of eukaryon PKlight, pee6.4 (preservation of this laboratory), IRES-EGFP, pee12.4 (are purchased from U.S. Clontech), and anti-EGFR resists Body LC, HC genetic fragment (preservation of this laboratory).

The clone of embodiment 1, hEGFR heavy chain of antibody and light chain gene：

Using the plasmid of the full-length gene containing anti-hEGFR as template, using pl1, pl2, ph1, ph2 two to primer respectively into The PCR amplification of row antibody light chain and heavy chain gene.

PCR amplification light chain reaction system (25 μ L systems) is as follows：

PCR amplification heavy chain reaction system (25 μ L systems) is as follows：

Each component carries out PCR reactions after mixing, and Amplification is：95 DEG C of pre-degeneration 7min, 95 DEG C of 45s, 60 DEG C 30s, 72 DEG C of 45s, after 15 circulations, 72 DEG C extend 10min eventually.After amplification, take under 2 μ L PCR product 120V voltages into Row agarose gel electrophoresis, observe clip size, and the result is shown in Fig. 2, as figure shows, carries out the PCR of antibody light chain and heavy chain gene Amplification, respectively obtains the specific purpose band of about 650bp and 1 400bp sizes, with expected 648bp and 1350bp sizes It is consistent, primarily determines that as LC, HC gene.

Embodiment 2, the purifying of PCR product and the connection with cloning vector

The correct pcr amplification product of molecular weight (concentration is about 100ng/ μ L) 20 μ L are taken to carry out 1% Ago-Gel respectively Electrophoresis recycles, and voltage is set as 80V.Glue reclaim step is with reference to OMEGA plastic recovery kit specifications, and the result is shown in Fig. 3.Will purifying Product afterwards respectively takes 1 μ L to carry out electrophoresis, observes purification result.By light chain after purification and heavy chain respectively with pMD18-T simple Cloning vector is connected, and linked system (10 μ L systems) is as follows：

PMD18-T simple 1ul

The PCR product 4ul of purifying

Solution I 5ul

After mixing, connected overnight in 16 DEG C of waters bath with thermostatic control.

Embodiment 3, the conversion of recombinant cloning vector and the extraction of plasmid

The connection product of gained is transformed into E.coil DH5 α competent cells, the LB tablets completed will be converted in 37 12~14h is cultivated under conditions of DEG C.Detailed process is as follows：

The competent cell that three pipes freeze is taken out, is melted on ice.By the connection product of light chain, heavy chain and pMD18-T carriers Each 5 μ L are separately added into wherein two pipe competent cells, are named as LC-T and HC-T.Another pipe is not added with any material as negative Control.After 25~35min of ice bath, 42 DEG C of water-bath heat shock 1min, in dislocation ice, place 2min.The 300 sterile LB of μ L are often added in pipe Fluid nutrient medium, is respectively coated on the LB solid plate culture mediums containing 100 μ g/mL Amp in 37 DEG C of shaking tables after modest recovery 1h On, 37 DEG C of 12~14h of constant temperature incubation.

The single bacterium colony that random each picking 3 is of moderate size from two pieces of tablets of LC-T and HC-T, is inoculated into containing 100 respectively In the 10mL LB fluid nutrient mediums of μ g/mL Amp, with overnight incubation under conditions of 220rpm, 37 DEG C in constant-temperature table.Collect Thalline, Plasmid DNA small extraction reagent kit specification of the plasmid extraction step with reference to OMEGA companies.

The identification of embodiment 4, positive recombinant

LC-T recombinant plasmids carry out digestion identification with restriction enzyme Bgl II/EcoR I, while do PCR identifications.

Bgl II, EcoR I digestion identification systems are as follows：

Totally 10 μ L systems, 37 DEG C of water-bath digestion 2h.1% agarose gel electrophoresis observes result.

PCR identification systems are as follows：

Totally 10 μ L systems, loop parameter is the same as embodiment 1.1% agarose gel electrophoresis observes result.

HC-T recombinant plasmids carry out double digestion identification with restriction enzyme BamH I, Nhe I, using 10 × H Buffer, digestion system are same as above.Primer Ph1 and Ph2 are applied in the PCR identifications of HC-T recombinant plasmids, and identification system is same as above, circulation ginseng Number is the same as embodiment 1.The result is shown in Fig. 4, understands that positive recombinant plasmid LC-T carries out double digestion identification with Bgl II/EcoR I from figure, Two bar segments, respectively the antibody light chain base of the pMD18-T simple carriers of about 2 700bp and about 650bp are obtained after digestion Cause, is consistent with expected stripe size.Using pl1, pl2 as primer, recombinant plasmid LC-T is template, carries out PCR identifications, obtains one The band of treaty 650bp, is consistent with expected 648bp sizes.Positive recombinant plasmid HC-T carries out double enzymes with BamH I/Nhe I Identification is cut, obtains two bar segments, respectively the heavy chain of antibody base of the pMD18-T simple carriers of about 2 700bp and about 1 400bp Cause, is consistent with expected stripe size.Using ph1, ph2 as primer, recombinant plasmid HC-T is template, carries out PCR identifications, obtains one The band of 1 400bp of treaty, is consistent with expected 1 350bp sizes.

Embodiment 5, hEGFR antibody light chains, the measure of heavy chain gene sequences and analysis

PCR, digestion are identified that all correct light chain and heavy chain recombinant plasmid carry out sequencing by Shanghai Ying Jun companies.Adopt The sequencing result of gene is analyzed with DNAMAN biological softwares.The results show：Clone the gene open reading frame obtained Respectively 648bp and 1 350bp, is separately encoded 215 and 449 amino acid, non-producer mutation.

It is prepared by the digestion of embodiment 6, LC, HC, IRES EGFP gene fragment and pKlight, pee12.4, pee6.4 carrier

To be sequenced pee12.4-IRES EGFP, pKlight that correct LC-T, HC-T and laboratory preserve, In pee12.4, pee6.4 conversion Escherichia coli E.coil DH5 α competent cells, the next day difference single bacterium colony of picking, and be inoculated with In the 10mL LB fluid nutrient mediums containing 100 μ g/mL Amp, to be cultivated under conditions of 220rpm, 37 DEG C in constant-temperature table Plasmid, Plasmid DNA small extraction reagent kit specification of the plasmid extraction step with reference to OMEGA companies are extracted after 10~12h.

LC-T is handled with Bgl II/EcoR I double digestions, prepare LC genetic fragments (Bgl II and BamH I are isocaudarner).

Bgl II/EcoR I double digestion systems are as follows：

Totally 100 μ L systems, 37 DEG C of water-bath digestion 3h.

HC-T and pKlight carriers are handled respectively with BamH I/Nhe I double digestions, using 10 × M buffer；BamH I/EcoR I double digestions handle pKlight carriers, 10 × K buffer；Hind III/EcoR I double digestions are handled respectively Pee12.4 and pee6.4 carriers, 10 × M buffer；It is prepared by Nhe I/EcoR I double digestions processing pee12.4-IRES EGFP IRES EGFP fragments, 10 × M buffer.All digestion systems are same as above.

Above-mentioned all digestion products with 1% agarose gel electrophoresis be separately recovered LC, HC, IRES EGFP fragment and PKlight, pee12.4 and pee6.4 carrier, voltage are set as 80V.Glue reclaim step is with reference to OMEGA plastic recovery kit explanations Book.

The result is shown in Fig. 5, it is known that handles recombinant vector LC-T with Bgl II/EcoR I double digestions, glue reclaim obtains about 650bp LC fragments, be consistent with expected size；Recombinant vector HC-T and pKlight are handled respectively with BamH I/Nhe I double digestions to carry Body, glue reclaim respectively obtain the HC fragments of about 1 400bp and the pKlight carrier segments of about 6500bp, as a result with being expected greatly It is small to be consistent.PKlight carriers are handled with BamH I/EcoR I double digestions, glue reclaim obtains the pKlight carriers of about 6 300bp Fragment, is consistent with expected size；Hind III/EcoR I double digestions handle pee12.4 and pee6.4 carriers, glue reclaim point respectively The carrier segments for not obtaining about 7 500bp and about 5 000bp are consistent with expected size.

Embodiment 7, Pee12.4-LC, the structure of pee6.4-HC-IRES EGFP recombinant vectors

By LC (the Bgl II/EcoR I) fragments after recycling and pKlight (BamH I/EcoR I) carrier, HC (BamH I/Nhe I) fragment is connected with pKlight (BamH I/Nhe I) carrier with T4DNA ligases, and system (10 μ L) is as follows：

The product that LC, HC and pKlight carrier connect is transformed into bacillus coli DH 5 alpha respectively, extraction plasmid pK-LC, PK-HC, Plasmid DNA small extraction reagent kit specification of the extraction step with reference to OMEGA companies.Hind III/ are used after extraction plasmid EcoR I double digestion pK-LC and pK-HC carrier recoveries contain the fragment of IgKappa Leader, IgK-LC (Hind III/EcoR I)、IgK-HC(Hind III/EcoR I).37 °C of digestion 3h, system (100 μ L) are as follows：

Glue reclaim fragment IgK-LC (Hind III/EcoR I), IgK-HC (Hind III/EcoR I), concrete operations step Rapid reference OMEGA plastic recovery kit specifications.Recycling uses the mono- enzymes of Nhe I again after obtaining IgK-HC (Hind III/EcoR I) Cut to obtain IgK-HC (Hind III/Nhe I).37 DEG C of digestion 3h, system (50 μ L) are as follows：

Glue reclaim fragment IgK-HC (Hind III/Nhe I) and with IRES-EGFP (Nhe I/EcoR I), pEE6.4 (Hind III/Nhe I) three fragments of carrier are connected.IgK-LC (Hind III/EcoR I) and pEE12.4 (Hind III/ EcoR I) carrier is connected.Overnight, linked system (10 μ L) is as follows for 16 DEG C of connections：

Connection product is converted into bacillus coli DH 5 alpha respectively, extraction plasmid Pee12.4-LC, Pee6.4-HC-IRES- EGFP, Plasmid DNA small extraction reagent kit specification of the extraction step with reference to OMEGA companies.

The result is shown in Fig. 6：The digestion identification and PCR identifications of pK-LC, pK-HC recombinant plasmid, it is known that with Hind III/EcoR I double digestions identify pK-LC recombinant plasmids, and the LC fragments and two sizes for obtaining the Leader containing IgKappa of about 710bp are about 5 The pKlight carrier segments of 000bp and 1 200bp；PK-HC recombinant plasmids are identified with BamH I/Nhe I double digestions, respectively To about 6 500bp pKlight carrier segments and about 1 350bp the Leader containing IgKappa HC fragments, obtain two kinds of pieces Section is consistent with expected size.PCR mirror are carried out to recombinant vector pK-LC and pK-HC using primer pl1, pl2 and ph1, ph2 respectively It is fixed, the LC fragments of a treaty 650bp and the HC fragments of about 1 400bp are finally obtained, is consistent with expected size.Tentatively confirm as PK-LC, pK-HC are built successfully.

The result is shown in Fig. 7：The double digestion product recycling result of each recombinant plasmid.Understand with Hind III/EcoR I to pK-LC Double digestion is carried out, recycles the fragment containing LC, pee12.4-IRES EGFP is handled with Nhe I/EcoR I double digestions, obtains about 1 The IRES EGFP fragments of 300bp；Double digestion is carried out to pK-HC with Hind III/EcoR I, recycling contains HC (Hind III/ EcoR I) fragment, then contain with Nhe I single endonuclease digestions the fragment of HC, recycling HC (Hind III/Nhe I).

Result figure 8：Pee12.4-LC, pee6.4-HC-IRES EGFP digestions and PCR identifications.Hind III/ are used respectively EcoR I carry out double digestion identification to pee12.4-LC, pee6.4-HC-IRES EGFP, are obtained about after pee12.4-LC digestions The LC fragments of the pEE12.4 carrier segments of 7 500bp and the Leader containing IgKappa of about 710bp, size are consistent with expection； Obtain that fragment is descending to be successively after pee6.4-HC-IRES EGFP digestions：The pEE6.4 carrier segments of about 5 000bp, about 1 The Ig-HC (the HC fragments of the Leader containing IgKappa) of the upstream portion containing IRES of 600bp, the downstream containing IRES of about 1 070bp Partial EGFP fragments, each clip size are consistent with expection.Respectively using primer pl1, pl2 and ph1, ph2 to recombinant vector Pee12.4-LC, pee6.4-HC-IRES EGFP carry out PCR identifications, finally obtain the LC fragments and about 1 of a treaty 650bp The HC fragments of 400bp, are consistent with expected size.Primarily determine that recombinant plasmid pee12.4-LC, pee6.4-HC-IRES EGFP structures Build up work(.

The structure of embodiment 8, Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon

It will identify that correct Pee12.4-LC, Pee6.4-HC-IRES-EGFP plasmid carries out double enzymes with Not I/Sal I Cut.37 DEG C of digestion 3h, system (100 μ L) are as follows：

Glue reclaim contains the fragment of LC and HC-IRES-EGFP, is connected with T4DNA ligases, linked system (10 μ L) is such as Under：

Final connection product is converted into bacillus coli DH 5 alpha, peedual-IRES-EGFP-hEGFR plasmids is extracted, carries Take Plasmid DNA small extraction reagent kit specification of the step extraction step with reference to OMEGA companies.Not I/Sal I are used after extraction plasmid Double digestion identification is carried out, while double digestion identification is carried out with Hind III/EcoR I, size is shown in the expected experimental result that is consistent Fig. 9.Illustrate the success of peedual-IRES-EGFP-hEGFR construction of eukaryotic expression vector.

Embodiment 9, the extracting for removing endotoxic peedual-IRES-EGFP-hEGFR plasmids and concentration mensuration

E.coil DH5 α positive bacterium solutions containing peedual-IRES-EGFP-hEGFR plasmids are inoculated in containing 100 μ g/ In the 20mL LB culture mediums of mL Amp, 220rpm, 37 DEG C of 12~14h of constant-temperature table culture activation positive bacterias, then will activate Bacterium solution be all inoculated in the LB culture mediums that 400mL contains 100 μ g/mL Amp 8~12h of activation.Thalline is collected, according to OMEGA companies go endotoxin plasmid extraction kit specification to be stripped plasmid, by institute's upgrading grain uv-spectrophotometric Meter (ND 1000) measured concentration is simultaneously stored in -20 DEG C of refrigerators, for cell transfecting.

Embodiment 10, the electricity of peedual-IRES-EGFP-hEGFR plasmids turns and the expression analysis of anti-hEGFR

Take the logarithm growth period CHO-K1 cells with 1:3 concentration passage, in 37 DEG C, 5%CO2 incubators culture until Degree of converging is 70~80%, and with 0.25% Trypsin Induced, cell is washed once with PBS after centrifuging 3min with 1 000r/min, Cell is diluted with serum free medium and is counted, makes its concentration up to 1 × 10⁷A/mL.Take after purification and without endotoxic 20 μ g of peedual-IRES-EGFP-hEGFR Plasmid DNA are added in 400 μ L cell suspensions, 250V, 1 000 Ω, 1 000 μ F ginsengs Several lower electricity that carry out turn.Cell after transfection is transferred in the complete medium containing 10%FBS culture of recovering.

Screening and culturing medium of the replacement containing 50 μM of MSX carries out pressurization culture after electricity turns 24-30h, is only transferred to the thin of the carrier Born of the same parents can just survive, and positive cell observation is carried out using fluorescence inverted microscope in pressure process.Collect cells and supernatant into The expression analysis (Figure 10) of row anti-hEGFR.

Sequence table

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Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

agt gga tca ggg aca gat ttt act ctt agc atc aac agt gtg gag tct 240

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

gaa gat att gca gat tat tac tgt caa caa aat aat aac tgg cca acc 288

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

acg ttc ggt gct ggc acc aag ctg gag ctg aag cga act gtg gct gca 336

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

cca tct gtc ttc atc ttc cct cca tct gat gag cag ttg aaa tct gga 384

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

aaa gtg cag tgg aag gtg gat aac gcc ctc caa tcc ggt aac tcc cag 480

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

gcc tgc gaa gtc acc cat cag ggc ctg agc tct ccc gtc aca aag agc 624

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

ttc aac agg gga gag tgt tga 645

Phe Asn Arg Gly Glu Cys

210

<210> 5

<211> 214

<212> PRT

<213> Artifical sequence

<400> 5

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 6

<211> 1350

<212> DNA

<213> Artifical sequence

<220>

<221> CDS

<222> (1)..(1350)

<400> 6

cag gtg cag ctg aag cag tca gga cct ggc ctc gtg cag ccc tca cag 48

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

agc ctg tcc atc acc tgc aca gtc tct ggt ttc tca tta act aac tat 96

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

ggt gta cac tgg gtt cgc cag tct cca gga aag ggt ctg gag tgg ctg 144

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

gga gtg ata tgg agt ggt gga aac aca gac tat aat aca cct ttc aca 192

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

tcc aga ctg agc atc aac aag gac aat tcc aag agc caa gtt ttc ttt 240

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

aaa atg aac agt ctg caa tct aat gac aca gcc ata tat tac tgt gcc 288

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

aga gcc ctc acc tac tat gat tac gag ttt gct tac tgg ggc caa ggg 336

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

act ctg gtc act gtc tct gca gcc tcc acc aag ggc cca tcc gtc ttc 384

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcc gcc ctg 432

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

ggc tgc ctg gtc aag gac tac ttc ccc gaa cct gtg acg gtg tcc tgg 480

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc ctc 528

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc 576

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc 624

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

agc aac acc aag gtg gac aag cgc gtt gag ccc aaa tct tgt gac aaa 672

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

act cac aca tgc cca cct tgc cca gca cct gaa ctc ctg ggg gga cct 720

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc 768

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac 816

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat 864

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

gcc aag aca aag cct cgg gag gag cag tac aac agc acg tac cgg gtg 912

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag 960

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa 1008

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc 1056

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc agc ctg acc 1104

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag 1152

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

agc aat ggg cag ccc gag aac aac tac aag acc acg cct ccc gtg ctg 1200

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

gac tcc gac ggc tcc ttc ttc ctc tat agc aag ctc acc gtg gac aag 1248

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag 1296

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccc ggt 1344

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

aaa tga 1350

Lys

<210> 7

<211> 457

<212> PRT

<213> Artifical sequence

<400> 7

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Glu Thr Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

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

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Glu Thr Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Glu Thr Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met Glu Thr His Glu Ala Leu His Asn His Tyr Thr Gln

435 440 445

Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

<210> 8

<211> 60

<212> DNA

<213> Artifical sequence

<220>

<221> CDS

<222> (1)..(60)

<400> 8

gct cga gcc acc atg gag aca gac aca ctc ctg cta tgg gta ctg ctg 48

Ala Arg Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu

1 5 10 15

ctc tgg gtt cca 60

Leu Trp Val Pro

<210> 9

<211> 20

<212> PRT

<213> Artifical sequence

<400> 9

Ala Arg Ala Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu

1 5 10 15

Leu Trp Val Pro

20

Claims

A kind of 1. construction of eukaryotic expression vector method of high efficient expression anti-hEGFR genes, it is characterised in that following steps：

(1) clone of hEGFR heavy chain of antibody and light chain gene：

Using the plasmid of the full-length gene containing anti-hEGFR as template, design primer pl1, pl2, ph1, ph2 two to primer respectively into The PCR amplification of row full length antibody light chain and heavy chain gene；

(2) structure of Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon：

Light chain and heavy chain are inserted into pKlight carriers, obtain plasmid pK-LC and pK-HC.PK-LC plasmids are connected after digestion It is connected in pee12.4 carrier for expression of eukaryon；PK-HC plasmids and IRES EGFP are connected to pee6.4 carriers after digestion.Restructuring Pee6.4 and pee12.4 carriers afterwards use Sal I/Not I double digestions, recycle large fragment and are allowed to connected, construct at the same time Dicistronic expresssion plasmid vector with antibody light chain and heavy chain, is named as peedual-IRES-EGFP-hEGFR；

(3) endotoxic peedual-IRES-EGFP-hEGFR plasmids are removed in extraction.
2. the clone of the hEGFR heavy chain of antibody and light chain gene according to claim 1 step (1), it is characterised in that：Design Primer is
3. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：When connecting light chain and pKlight carriers, first T4 is used after restriction enzyme Bgl II, EcoR I respectively digestion Ligase connects, and obtains plasmid pK-LC.
4. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：When connecting heavy chain and pKlight carriers, first connected after restriction enzyme BamH I, Nhe I distinguish digestion with T4 Enzyme connection is connect, obtains plasmid pK-HC.
5. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：Contain IgKappa Leader in pKlight carriers.IgKappa Leader coding gene sequences are SEQ ID NO:Shown in 8, amino acid sequence is SEQ ID NO:Shown in 9.
6. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：PK-LC is connected after Hind III, EcoRI digestions obtain the light chain segments IgK-LC of the Leader containing IgKappa It is connected in pee12.4 carrier for expression of eukaryon, obtains plasmid Pee12.4-LC.
7. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：By pK-HC plasmids after Hind III/EcoR I double digestions, the heavy chain fragment of the Leader containing IgKappa is obtained IgK-HC。
8. the structure of Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 7, its feature exist In：IgK-HC is used to subsequently connect with fragment IgK-HC (Hind III/Nhe I) is obtained after Nhe I single endonuclease digestions.
9. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：Pee12.4 carriers and pee6.4 carriers are used to connect after Hind III, EcoRI digestions processing.
10. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：IRES EGFP fragments are prepared with Nhe I/EcoR I double digestions processing pee12.4-IRES EGFP, for carrier Structure.IRES EGFP fragment-encoding genes sequence is SEQ ID NO:Shown in 3.
11. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：IgK-HC (Hind III/Nhe I) is connected to obtain plasmid Pee6.4-HC- with IRES EGFP fragments, pEE6.4 IRES-EGFP。
12. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：Connect, obtain after Pee12.4-LC, Pee6.4-HC-IRES-EGFP plasmid are carried out double digestion with Not I/Sal I To plasmid Peedual-IRES-EGFP-hEGFR.Wherein LC coding gene sequences are SEQ ID NO:Shown in 4, amino acid sequence For SEQ ID NO:Shown in 5；HC coding gene sequences are SEQ ID NO:Shown in 6, amino acid sequence is SEQ ID NO:7 institutes Show.
13. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：Internal ribosome entry site (IRES) is introduced between heavy chain gene and EGFP gene, IRES is from brain cardiac muscle Scorching virus, it is functionally similar to the 5' cap sequences on eukaryotic mRNA, mainly recruits ribosomes and enters and make its upstream And two transcriptons in downstream are translated at the same time.
14. the structure of the Peedual-IRES-EGFP-hEGFR carrier for expression of eukaryon according to claim 1 step (2), its It is characterized in that：Enhanced green fluorescence protein (EGFP) gene is introduced in the downstream of light chain, intuitively can monitor and sort sun Property cell.Wherein EGFP coding gene sequences are SEQ ID NO:Shown in 1, amino acid sequence is SEQ ID NO:Shown in 2.