CN112961865B

CN112961865B - Recombinant trastuzumab expressed by gene editing chicken bioreactor and application

Info

Publication number: CN112961865B
Application number: CN202110198516.1A
Authority: CN
Inventors: 唐小川; 梁晶晶; 魏姣; 梁瑞益
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2021-02-22
Filing date: 2021-02-22
Publication date: 2023-06-02
Anticipated expiration: 2041-02-22
Also published as: CN112961865A

Abstract

A recombinant trastuzumab expressed by a gene editing chicken bioreactor and application thereof belong to the technical field of genetic engineering. The nucleotide sequence is shown in a sequence table No.1, and the two expressed amino acid sequences are shown in sequence tables No.2 and No. 3. Constructing a gene knock-in vector containing the gene according to a CRISPR/Cas9 gene editing system; screening high-efficiency CRISPR/Cas9 knock-in sites at chicken ovalbumin gene start sites; microinjection is carried out on chicken embryo blood vessels which develop to 2.5 days by using an adenovirus vector packaging gene editing tool and the knock-in vector, so as to realize the fixed-point knock-in of trastuzumab genes in the knock-in site; after the chick embryo is hatched and grown up, the trastuzumab is expressed in a large quantity and secreted into eggs, so that the efficient production of the trastuzumab is realized. The trastuzumab antibiotic production method has the advantages of low cost and high product activity, and can effectively inhibit the growth of the breast cancer cell line cultured in vitro.

Description

Recombinant trastuzumab expressed by gene editing chicken bioreactor and application

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a recombinant trastuzumab expressed by a gene editing chicken bioreactor and application thereof.

Background

Trastuzumab (Trastuzumab) is a monoclonal antibody targeting epidermal growth factor receptor 2 (HER 2), and the trade name Herceptin (Herceptin) is one of the most effective drugs for the treatment of breast cancer.

Breast cancer is one of the most common malignant tumors of women, is also a common cause of death of the tumors of the women, and seriously threatens the body health of vast women. In 1987, slamon et al, in a 189-case study of human breast cancer, reported for the first time the amplification of the protooncogene HER2, and indicated that tumors were susceptible to recurrence and that clinical prognosis were poor due to the multicopy nature of this gene. In 1997, graus-Porta et al studied the correlation of HER2 with other oncogenes of the HER family, suggesting that HER2 is a theoretical model of growth factor receptor, revealing that HER2 is an oncogene. This gene is thought to be overexpressed in tumors, whereas in normal tissues the expression levels are very low, HER2 being identified as a new tumor marker. Several studies confirm the relevance of HER2 to breast cancer, and have now become recognized important molecular markers and therapeutic targets for breast cancer, and studies indicate that it plays an important role in regulating cell growth, differentiation, adhesion and affecting survival rate. The data of preclinical and clinical studies show that trastuzumab directed against HER2 extracellular region can be used for postoperative adjuvant treatment of HER2 high-expression breast cancer patients, and treatment of HER2 high-expression metastatic breast cancer patients, 26 cases of trastuzumab treatment of HER2 positive metastatic breast cancer have remarkable curative effects, the recent remission rate is related to HER2 expression conditions, and the occurrence rate of adverse reactions is low. In addition, trastuzumab and chemotherapy have remarkable effects in treating breast cancer.

However, trastuzumab is mainly produced by culturing mammalian cells on a large scale at present, and has the defects of high production cost, small yield and the like, so that the trastuzumab has high price and high clinical treatment cost, and the cost of one treatment course is 3.2 ten thousand dollars. Trastuzumab is in great demand and sold in the world in 2014 for 55.64 billion dollars. Although several research units in China can produce trastuzumab at present, the yield and activity of trastuzumab are limited, and the trastuzumab cannot be applied to clinic on a large scale.

With the development and popularization of gene editing technology, the production of recombinant protein drugs by using living gene editing animals, namely, a gene editing animal bioreactor, has become possible. The production method has great advantages in the aspects of cost, product activity, safety and the like.

Disclosure of Invention

In order to overcome the defects of the prior art for producing trastuzumab, the invention provides recombinant trastuzumab expressed by utilizing a gene editing chicken bioreactor and application thereof by utilizing the advantages of the fixed-point knock-in type gene editing chicken, constructs a fixed-point knock-in expression vector containing the gene, and utilizes the vector and the CRISPR/Cas9 gene editing vector to produce the recombinant trastuzumab gene to be efficiently inserted into egg white protein sites of chicken at fixed points so as to realize efficient expression and secretion of trastuzumab in egg white.

According to the wild trastuzumab sequence, the gene sequence is redesigned according to the preferred codons of the chickens, and the sequence is different from the wild trastuzumab sequence to a certain extent.

A nucleotide sequence of the recombinant trastuzumab gene expressed by the gene editing chicken bioreactor is shown in a sequence table No. 1.

The amino acid sequences of the two sections of proteins coded by the genes are shown in sequence tables No.2 and No. 3.

The application of the protein in preparing medicaments for treating breast cancer.

And a CRISPR/Cas9 gene editing site which is suitable for carrying out site-specific knock-in by utilizing a knock-in vector of a recombinant trastuzumab gene expressed by a gene editing chicken bioreactor, wherein the site is positioned on a second exon of a chicken ovalbumin gene, and the sequence of the site is shown as a sequence table No. 4.

The method for editing the recombinant trastuzumab gene expressed by the chicken bioreactor by using the genes comprises the following steps:

(1) Packaging adenovirus vectors: packaging site-directed knock-in vector and vector comprising sequence of CRISPR/Cas9 gene editing tool into adenovirus vector with high infection capability, and titer of the adenovirus vector is not less than 1×10 ¹⁰ PFU/mL；

(2) Microinjection of chick embryo: injecting the adenovirus vector into a chicken embryo blood vessel hatched for 2.5 days by a microinjection technology, continuously hatching the chicken embryo until the chicken embryo is out of the shell, and feeding the chicken embryo until sexual maturity;

(3) Product concentration and activity detection: the successfully fixed-point knocked-in gene editing chicken is fed in a large quantity and propagated, a cock is reserved as a breeder, hen collects eggs, egg white is separated, the concentration of recombinant trastuzumab is measured through ELISA (Enzyme-linked immunosorbent assay ), human breast cancer cells are cultured in vitro, and the produced trastuzumab is added into a culture medium to detect the capacity of inhibiting proliferation of the breast cancer cells.

Wherein, the fixed-point knock-in type gene editing chicken identification: detecting chicken semen by PCR (Polymerase chain reaction ), if the chicken semen is positive, namely, the chicken semen is used as a breeding chicken for breeding, and the produced PCR positive offspring chicken is the fixed-point knock-in type gene editing chicken;

the invention constructs the knock-in carrier of trastuzumab gene, takes the adenovirus carrier as a basic skeleton, can be used for packaging adenovirus, produces adenovirus carrier with high titer and cell infection function, uses the adenovirus carrier to inject and develop chicken embryo for 2.5 days, enables the adenovirus to infect germ cells of the chicken embryo, and can efficiently produce gene knock-in gene editing chicken.

In order to enable the knock-in vector to be knocked in to a designated chicken ovalbumin site at a fixed point after entering chicken germ cells, the constructed knock-in vector comprises two targeting sequences which are 500bp in length and homologous to the chicken ovalbumin nucleotide sequence. The CRISPR/Cas9 is utilized to carry out fixed-point cutting on chicken ovalbumin, two sections of homologous sequences positioned at the upstream and downstream of the cutting site can utilize the self-homologous repair function of cells, and the recombinant trastuzumab gene sequence positioned inside the two sections of homologous sequences is inserted into the cutting site at fixed points, so that fixed-point knock-in is realized.

In order to enable the trastuzumab gene sequence knocked in at fixed points to be expressed normally, the 2A connecting peptide sequence is added in front of the gene sequence, and the sequence has the functions of connecting the trastuzumab gene sequence with the residual ovalbumin sequence, and realizing the expression of trastuzumab while efficiently expressing the residual ovalbumin by utilizing an ovalbumin promoter. The 2A linked peptide is degraded by hydrolytic enzymes within the cell after gene expression and protein translation are completed, thereby releasing the complete trastuzumab from the residual ovalbumin.

In order to enable trastuzumab expressed in chicken cells to be secreted out of the cells into egg white more efficiently, the signal peptide sequence of trastuzumab protein is changed into chicken lytic enzyme signal peptide, and the signal peptide can be used for guiding the trastuzumab to be secreted into egg white efficiently because lysozyme is one of main protein components of egg white, and the signal peptide can be cut in the secretion process and cannot remain on the trastuzumab. The design ensures that all trastuzumab synthesized in the fixed-point knock-in type gene editing chicken body is collected into egg white, so that on one hand, the yield and purity of a product are increased, and on the other hand, the toxic and side effects of the product on the chicken are reduced.

As a further improvement of the invention, a segment of PolyA sequence for stopping gene expression is connected to the downstream of the recombinant trastuzumab sequence in the knock-in vector, which has the function of ensuring that the residual C-terminal fragment of the ovalbumin is not expressed after the trastuzumab is expressed, and the residual C-terminal fragment of the ovalbumin is positioned at the downstream of the trastuzumab and cannot be translated due to the stop codon of the trastuzumab in the protein translation process, but the residual amino acid residue number is more, so that the expression amount of the trastuzumab can be reduced to a certain extent. It should be noted that the addition of PolyA sequences also increases the length of the entire knock-in fragment, thereby reducing the efficiency of gene knockin, and therefore whether or not to add PolyA sequences to a knock-in vector will depend on the specific efficiency of gene knockin.

The invention finds a most efficient site on ovalbumin, which is suitable for cutting CRISPR/Cas9 and knocking in a carrier for fixed-point knocking in. The locus is positioned on a second exon of the ovalbumin gene, and the sequence of the locus is shown as a sequence table No. 4. The procedure for determining this site is as follows: firstly, carrying out predictive analysis on a target area through on-line software (https:// www.benchling.com/crispr /), and preliminarily determining 4 alternative sites; then, chicken embryo fibroblasts are cultured in vitro, transfection is carried out by using CRISPR/Cas9 transient expression vectors targeting the 4 sites, CRISPR/Cas9 gene editing efficiency detection is carried out by using a T7E1 enzyme digestion method, and the final knock-in site is the highest detection positive rate.

As a further optimization of the present invention, an adenovirus vector titer detection step can be added between the step (1) and the step (2). The process is summarized as follows: culturing human kidney epithelial cell line (293T cell) in vitro, and plating in 48-hole culture dish after the growth curve enters exponential phase; when the confluency of 293T cells after being plated is about 70%, carrying out gradient dilution and infection by using the packaged adenovirus vector; the infected cells are continuously cultured, the infection rate of the adenovirus vector is judged according to the plaque condition on the cells, and the titer is calculated according to the infection rate and the dilution factor. Only ensuring that adenovirus vector titres are not less than 1X 10 ¹⁰ After PFU/mL, a subsequent chick embryo microinjection step can be performed, otherwise positive fixed-point knock-in gene editing chicks are difficult to obtain.

As a further optimization of the present invention, if the final packaged adenovirus vector titer cannot reach 1X 10 ¹⁰ PFU/mL, can be modified in the step (2), namely, after separating the primordial germ cells in the blood of the 2.5-day chick embryo, the primordial germ cells infected with the adenovirus vector are infected in vitro, and then the primordial germ cells infected with the adenovirus vector are injected into new chick embryo. The improvement can improve the infection efficiency of adenovirus vector to germ cells, but has the defect that the separation of the original germ cells in blood is a time-consuming and labor-consuming step, and can ensure that the packaged virus titer reaches 1X 10 ¹⁰ When PFU/mL or more, microinjection of chick embryos is preferably performed using the original protocol.

The invention has the beneficial effects that:

the fixed-point knock-in gene editing chicken of the invention optimizes target protein genes and constructs fixed-point knock-in vectors by utilizing a genetic engineering technology, and utilizes a third generation gene editing tool (CRISPR (Clustered regularly interspaced short palindromic repeats, regular indirect short palindromic sequence repeat cluster) technology at the forefront at present to lead exogenous genes to be directionally and stably integrated into chicken genome, so that exogenous proteins are efficiently expressed and secreted in egg white; the target protein can be obtained from egg white with low cost and high efficiency by breeding the gene-edited chicken and collecting a large amount of eggs.

Compared with the existing recombinant protein expression system, the method for producing the medicinal protein by using the gene editing chicken bioreactor has the following advantages:

1. the manufacturing is simple, and the early investment is less;

2. the yield efficiency is high, and the production period is short;

3. egg white is simple in component, low in purification cost and even free from purification in certain applications;

4. no protease, containing antibacterial enzyme, long protein stability half-life;

5. the glycosylation characteristics of the produced pharmaceutical protein are closest to those of human beings, and the biological activity is good;

6. green and environment-friendly, and has no chemical pollution and endotoxin.

Drawings

FIG. 1 is a flow chart of chick embryo microinjection.

Fig. 2 shows trastuzumab content in different numbered genetically edited egg white.

FIG. 3 shows the detection of the binding strength of trastuzumab to breast cancer cell membranes.

FIG. 4 is a graph showing the detection of growth inhibition of breast cancer cells by trastuzumab.

FIG. 5 shows trastuzumab apoptosis detection on breast cancer cells.

Detailed Description

Example 1

Knock-in vector construction comprising trastuzumab sequences.

(1) Cloning of upstream and downstream homology arms: two pairs of primers, named HR and HL, are synthesized respectively, and 30bp sequences homologous to the inserted fragments are additionally added to the 5' end of the downstream primer so as to fuse DNA fragments in the next step; the 5' end of the upstream primer is added with enzyme cutting sites BamHI and EcoRI so as to carry out enzyme cutting, and the chicken whole blood genome is used as a template for PCR amplification, and the amplification system is as follows:

the amplified product was electrophoresed using a 2% agarose gel, and then the 500bp band in the gel was recovered and purified using a gel recovery kit of Beijing Tiangen Biotechnology Co.

(2) In vitro synthesis comprising trastuzumab sequence insert: using software DNAstar, designing a chicken preferred trastuzumab gene sequence according to a chicken preference codon table and a wild trastuzumab amino acid sequence, adding a chicken lysozyme signal peptide sequence at the 5 'end of the sequence, and adding a 2A self-cleaving polypeptide sequence at the 3' end. The above sequences were submitted to chemical synthesis of gene sequences by Shanghai Biotechnology Co.

(3) Fragment fusion: the 3 fragments are fused by PCR, the principle is that the 3 fragments contain 30bp homologous sequences, primers (named RH) are designed at the 5 'end of an upstream homologous arm sequence and the 3' end of a downstream homologous arm sequence, and the fragments and the primers are mixed for amplification, wherein an amplification system is as follows:

the amplified product was electrophoresed using a 1.2% agarose gel, and then the 1340bp band in the gel was recovered and purified using a gel recovery kit of Beijing Tiangen Biotechnology Co.

(4) And (3) vector enzyme cutting and connection: the 1340bp fragment and adenovirus vector were digested with BamHI/EcoRI, and the digestion system was as follows:

after digestion at 37℃overnight, the mixture was purified using a DNA purification kit from Beijing Tiangen Biotechnology Co. The purified insert and the virus skeleton are subjected to a ligation reaction, and the reaction system is as follows:

after 6 hours of ligation at 16 degrees celsius, the reaction mixture was transformed into competent cells, which were plated on solid medium containing ampicillin. Single colony was picked up and cultured for 48 hours on a plate, and plasmid in the cultured bacterial liquid was extracted using a plasmid extraction kit from Beijing Tiangen Biotechnology Co.

(4) Identification of knock-in vector: the extracted plasmid was amplified by PCR using primers HL-F and HR-R, the amplification system was as in step (1), and the amplified product was subjected to gel electrophoresis and gel recovery, and then submitted to sequencing by Shanghai Biotechnology Co. If the sequencing result accords with the expected design, the vector is successfully constructed, and the vector is stored at minus 20 ℃.

Example 2

Microinjection of chick embryos was performed for 2.5 days.

The chick embryo microinjection process is shown in fig. 1, and the implementation details are described below:

(1) Purchasing hatching eggs in 12 hours of new production, cleaning with 0.1% of benzalkonium chloride, airing, and drawing a circle with the diameter of about 1.5 cm on the equatorial plane of the hatching eggs by using waterproof glue, wherein the inspection is carried out to ensure that the waterproof glue is airtight and water-proof; the eggs were incubated at 37.8 degrees celsius, 60% humidity, and once every hour at 90 degrees. Note that: if the hatching eggs are not hatched temporarily, the hatching eggs need to be stored in a refrigerator at 13 ℃ so as to prevent the hatching eggs from developing in advance; the waterproof glue is hardened for about 12 hours, eggs are horizontally laid and hatched before hardening, and the eggs can be hatched with big heads upwards after hardening.

(2) After hatching the hatching eggs for 2.5 days (55-60 hours, the period of chick embryo development was about HH 14-16), a hole of about 7 mm in diameter was made in the equatorial plane of the hatching eggs using a screw punch at 14000 rpm. Note that: punching only grinds off the calcified layers of eggshells, and the integrity of eggshell membranes is ensured; in order to prevent damage to the chick embryo in the punching process, the chick embryo is punched from the side or below, and the chick embryo floats right above the hatching eggs and is far away from the holes.

(3) Taking a plurality of common eggs, peeling eggshell membranes of the common eggs by using ophthalmic forceps, cutting the common eggs into square with a side length of about 1 cm by using ophthalmic scissors, and soaking the common eggs in Phosphate Buffer Solution (PBS) for standby. And (3) dripping a few drops of PBS (Poly vinyl acetate) into a ring formed by the sealant to isolate external air from entering into hatching eggs, then using a scalpel to scratch the eggshell membrane, and using ophthalmic forceps to take out the eggshell membrane, so that the chick embryo and peripheral blood vessels of the chick embryo can be observed. Note that: all the instruments and reagents used were autoclaved; the PBS dropped in the sealing rubber ring not only plays a role in isolating air and external pollutants, but also enables the chick embryo to float upwards to be close to the punched hole, and simultaneously plays an effect of a magnifying glass, thereby facilitating the microinjection of the next step.

(4) The adenovirus vector was removed from the minus 80 degree celsius refrigerator and slowly thawed in ice cubes at zero degrees. 2 microliters of the mixture was aspirated with a microinjection needle, and a reflux vessel in the peripheral vessel of the chick embryo was searched under a split microscope for microinjection. After the injection is finished, the eggshell membrane in the step (3) is used for covering holes on the egg, the PBS in the sealing rubber ring is sucked by the water absorbing paper, and the eggshell membrane for covering can be tightly stuck to the punched holes at the moment, so that air leakage can be avoided. And (3) sealing all the upper parts of the small holes by using new sealant, and placing the hatching eggs in an incubator for continuous hatching.

Example 3

Detecting the content of trastuzumab in egg white.

1. The detection was performed using a double antibody sandwich ELISA, the procedure of which is summarized as follows:

a. coating of antibodies murine anti-human trastuzumab antibody (primary antibody) was diluted 1:2000-fold with PBS containing 5% bovine serum albumin as coating solution, added to the ELISA plate at 100. Mu.l per well and coated overnight at 4 ℃.

b. The next day the plate was washed by pipetting up the Kong Nabao primary antibody solution using a pipetting gun, adding about 300 microliters of wash solution per well (PBS solution containing 5% detergent tween-20) for rinsing, rinsing 4 times at 100 revolutions per minute for 5 minutes on a shaker;

c. sealing, namely adding sealing liquid (PBS of 5% bovine serum albumin) into an ELISA plate according to 200 microliters of each hole, coating the ELISA plate on a shaking table at 50 revolutions per minute, wherein the temperature is 25 ℃ at room temperature for 2 hours, and sucking the sealing liquid in the holes by using a pipette after coating.

d. Diluting the sample and standard liquid, namely carrying out gradient dilution on egg white containing trastuzumab to be detected by using PBS (phosphate buffer solution), wherein each gradient dilution is 10 times, and total gradient is 12. Using a common egg white diluent as a negative control, diluting 3 gradients; commercial trastuzumab was used as a positive control, again diluted 3 gradients. Since egg white is more viscous, the initial dilution was 100-fold. The standard trastuzumab solution was diluted with PBS simultaneously for 10 gradients. The solutions were sequentially added to the ELISA plate at 100. Mu.l per well. The samples and standard solutions were incubated at room temperature for 2 hours at 25 degrees celsius.

e. After washing the plate according to step b, adding a 1:5000 dilution of goat anti-mouse antibody (enzyme-labeled secondary antibody) labeled with horseradish peroxidase to the ELISA plate according to 200 microliters per well. At each gradient. The solutions were sequentially added to the ELISA plate at 100. Mu.l per well. The samples and standard solutions were incubated at room temperature for 1 hour at 25 degrees celsius.

f. And d, after washing the plate according to the step b, adding a newly prepared color development liquid and TMB substrate into the ELISA plate according to 200 microliters per hole, and performing light-shielding reaction for 15 minutes at the room temperature of 25 ℃. Subsequently, 50. Mu.l of stop solution was added to each well.

g. The ELISA plate is placed in an ELISA apparatus and absorbance values at 450 nm are detected. Based on the absorbance values of the standards, an Excel software was used to make the standard curve. And taking the average value of the absorbance values of the egg white trastuzumab subjected to gradient dilution as the final concentration value.

h. The egg white samples were tested for three different batches, the average and standard deviation values were calculated. The concentration of trastuzumab in egg white is shown in fig. 2.

Example 4

Application of recombinant trastuzumab in inhibiting growth of cancer cells

The biological activity of recombinant trastuzumab was determined in vitro cultured HER2 positive breast cancer cell lines BT-474 and ZR-75-1 using commercial trastuzumab (from switzerland corporation, commercial herceptin) as an application control. The recombinant trastuzumab is incubated with a cancer cell line, fluorescent-labeled secondary antibody is added to bind the recombinant trastuzumab, the average fluorescence intensity on the cell membrane is analyzed by a flow cytometer to judge the binding condition of the recombinant trastuzumab and HER2 protein on the cell membrane of two breast cancer strains, and the result is shown in figure 3, in the concentration range of 0.01-10 mug/mL in the two breast cancer cell lines, the binding intensity of the recombinant trastuzumab prepared by the invention and the commercial trastuzumab is not obviously different from each other in the target protein.

The recombinant trastuzumab and the commercial trastuzumab prepared by the method are added into two breast cancer cell line culture media, and the results show that: the recombinant trastuzumab prepared in the present invention was not different from commercial trastuzumab in ZR-75-1 cells in terms of cell growth inhibition, and the recombinant trastuzumab prepared in the present invention was not different from commercial trastuzumab in the 0.1 μg/mL, 0.5 μg/mL, 1 μg/mL groups in BT-474 cells, whereas the cell growth inhibition effect of the recombinant trastuzumab prepared in the present invention was significantly higher than that of commercial trastuzumab in the 5 μg/mL, 10 μg/mL groups (FIG. 4). Flow cytometry analysis results after Annexin V staining show that 1 mug/mL, 5 mug/mL and 10 mug/mL of recombinant trastuzumab prepared by the invention in BT-474 cells can induce apoptosis more remarkably (figure 5).

Sequence listing

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

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Leu Ser Pro Gly Lys

465

<210> 1

<211> 19

<212> DNA

<213> Knock-in Site

<400> 1

ctctagccat ggtatacct 19

Claims

1. A recombinant trastuzumab gene expressed by utilizing a gene editing chicken bioreactor is characterized in that the nucleotide sequence of the recombinant trastuzumab gene is shown as a sequence table No. 1.

2. The two-segment protein coded by the gene as claimed in claim 1, wherein the amino acid sequences are shown in sequence tables No.2 and No.3, and are obtained by the following steps:

(1) Packaging adenovirus vectors: packaging site-directed knock-in vector and vector comprising sequence of CRISPR/Cas9 gene editing tool into adenovirus vector with high infection capability, and titer of the adenovirus vector is not less than 1×10 ¹⁰ The recombinant trastuzumab sequence contained in the adenovirus knock-in vector contains a 2A connecting peptide sequence and a chicken lytic enzyme signal peptide sequence, the gene editing site corresponding to the adenovirus CRISPR/Cas9 vector is positioned on the second exon of the chicken ovalbumin gene, and the sequence of the site is shown as a sequence table No. 4.

(3) Recombinant trastuzumab concentration and activity detection: and (3) raising and propagating a large number of successfully fixed-point knocked-in gene editing chickens, reserving cock as breeder chickens, collecting eggs of the cock chickens, separating egg white of the hen chickens, detecting the concentration and activity of recombinant trastuzumab, and purifying the egg white detected positive to obtain target proteins.

3. Use of the protein of claim 2 for the preparation of a medicament for the treatment of breast cancer.