CN112961865A

CN112961865A - Recombinant trastuzumab expressed by using gene editing chicken bioreactor and application

Info

Publication number: CN112961865A
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: 2021-06-15
Anticipated expiration: 2041-02-22
Also published as: CN112961865B

Abstract

A recombinant trastuzumab expressed by a chicken bioreactor by using gene editing and application thereof belong to the technical field of genetic engineering. The nucleotide sequence is shown in sequence table No.1, and the expression two sections of 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 a high-efficiency CRISPR/Cas9 knock-in site at the initiation site of the chicken ovalbumin gene; using an adenovirus vector packaging gene editing tool and the knock-in vector to carry out microinjection on the chick embryo blood vessel which develops for 2.5 days, so as to realize the fixed-point knock-in of the trastuzumab gene in the knock-in site; after the chick embryo is hatched and grows up, the trastuzumab is expressed in large quantity and secreted into the egg, so that the high-efficiency production of the trastuzumab is realized. The production method of trastuzumab 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 using gene editing chicken bioreactor and application

Technical Field

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

Background

Trastuzumab (Trastuzumab) is a monoclonal antibody targeting epidermal growth factor receptor 2(HER2), and is one of the most effective drugs for treating breast cancer under the trade name Herceptin (Herceptin).

The breast cancer is one of the most common malignant tumors of women and is also a common female tumor death reason, and the health of the majority of women is seriously threatened. In 1987, Slamon et al, in a 189 studies of human breast cancer, first reported the amplification of the proto-oncogene HER2, and indicated that tumors were susceptible to recurrence and poor clinical prognosis due to the multiple copies of this gene. In 1997, Graus-pora et al studied the interrelationship of HER2 with other oncogenes of the HER family, proposed a theoretical model for HER2 as a growth factor receptor, revealing HER2 as an oncogene. The gene is considered to be overexpressed in tumors, while the expression level in normal tissues is very low, and HER2 is identified as a novel tumor marker. The relevance of HER2 to breast cancer is proved by a plurality of researches, which become a recognized molecular marker and a therapeutic target for breast cancer at present, and the researches show that the HER2 plays an important role in regulating cell growth, differentiation, adhesion and influencing survival rate. The data of preclinical and clinical researches indicate that trastuzumab aiming at the extracellular region of HER2 can be used for postoperative adjuvant therapy of patients with HER2 high-expression breast cancer and treatment of patients with HER2 high-expression metastatic breast cancer, 26 cases of trastuzumab for treating HER2 positive metastatic breast cancer have remarkable curative effect, the recent remission rate is related to the HER2 expression condition, and the incidence rate of adverse reactions is low. In addition, trastuzumab has a remarkable effect on treating breast cancer by combining with chemistry.

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

With the development and popularization of gene editing technology, it is possible to produce recombinant protein drugs using living gene editing animals, i.e., a gene editing animal bioreactor. 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 utilizes the advantages of fixed-point knock-in type gene editing chicken, provides recombinant trastuzumab expressed by using a gene editing chicken bioreactor and application thereof, constructs a fixed-point knock-in expression vector containing the gene, and utilizes the vector and a CRISPR/Cas9 gene editing vector to produce a product capable of efficiently inserting the recombinant trastuzumab gene into an ovalbumin site of chicken at a fixed point, thereby realizing the efficient expression and secretion of the trastuzumab in egg white.

The invention redesigns the gene sequence according to the wild trastuzumab sequence and chicken preference codon, and the sequence has certain difference with the wild trastuzumab sequence.

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

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

The protein is applied to the preparation of medicaments for treating breast cancer.

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

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

(1) packaging an adenovirus vector: knocking the fixed pointThe vector and the vector containing the sequence of CRISPR/Cas9 gene editing tool are packaged into the adenovirus vector with high infection capacity, and the titer is not lower than 1 × 10¹⁰PFU/mL；

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

(3) and (3) detecting the concentration and the activity of a product: feeding and propagating a large number of the successfully knocked-in gene-edited chickens, reserving the cocks as breeding hens, collecting eggs of the hens, separating egg white of the hens, measuring the concentration of the recombinant trastuzumab by ELISA (Enzyme-linked immunosorbent assay), culturing human breast cancer cells in vitro, adding the produced trastuzumab into a culture medium, and detecting the capacity of the produced trastuzumab for inhibiting the proliferation of the breast cancer cells.

Wherein, identifying the chicken by fixed-point knock-in type gene editing: detecting the chicken semen by PCR (Polymerase chain reaction), and breeding the chicken as a breeding hen if the result is positive, wherein the produced PCR positive progeny chicken is a fixed-point knock-in type gene editing chicken;

the invention constructs a knock-in vector of trastuzumab gene, which takes an adenovirus vector as a basic skeleton, can be used for packaging adenovirus to produce the adenovirus vector with high titer and cell infection function, and the adenovirus vector is used for injecting chick embryo which grows to 2.5 days, so that the adenovirus infects germ cells of the chick embryo, and can efficiently produce gene knock-in type gene editing chick.

In order to ensure that the knock-in vector can be knocked into a specified egg white protein site at a fixed point after entering chicken germ cells, the constructed knock-in vector comprises two targeting sequences with the lengths of both 500bp and homologous nucleotide sequences of the egg white protein. The CRISPR/Cas9 is utilized to carry out site-directed cleavage on the egg albumin, the two homologous sequences positioned at the upstream and the downstream of the cleavage site can utilize the homologous repair function of cells to insert the recombinant trastuzumab gene sequence positioned in the two homologous sequences into the cleavage site in a site-directed manner, thereby realizing site-directed knock-in.

In order to ensure that the gene sequence of the trastuzumab knocked in at a fixed point can be normally expressed, a 2A connecting peptide sequence is added in front of the gene sequence, the sequence has the function of connecting the gene sequence of the trastuzumab with a residual ovalbumin sequence, and the expression of the trastuzumab is realized while the residual ovalbumin is efficiently expressed by utilizing an ovalbumin promoter. After gene expression and protein translation, the 2A connecting peptide can be degraded by hydrolytic enzyme in cells, so that the intact trastuzumab is released from residual egg albumin.

In order to enable trastuzumab expressed in chicken cells to be more efficiently secreted out of the cells and enter egg white, the signal peptide sequence of trastuzumab protein per se is changed into chicken lytic enzyme signal peptide, lysozyme is one of main protein components of the egg white, the lytic enzyme signal peptide can efficiently guide trastuzumab to be secreted into the egg white, and the signal peptide per se can be cut in the secretion process and cannot be remained on the trastuzumab. The design enables all trastuzumab synthesized in the body of the fixed-point knock-in type gene editing chicken to be gathered in egg white, so that the yield and the purity of the product are increased, and the toxic and side effects of the product on the chicken are reduced.

As a further improvement of the invention, a polyA sequence expressed by a termination gene is connected at the downstream of a recombinant trastuzumab sequence in a knock-in carrier, the action is to ensure that the residual C-terminal fragment of the redundant ovalbumin is not expressed after the trastuzumab is expressed, although the residual C-terminal fragment of the redundant ovalbumin is positioned at the downstream of the trastuzumab, the residual C-terminal fragment of the redundant ovalbumin cannot be translated due to the termination codon of the trastuzumab in the process of protein translation, but the quantity of the redundant amino acid residues is more, and the expression quantity 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 knock-in, and therefore whether PolyA sequences are added to the knock-in vector will depend on the specific efficiency of gene knock-in.

The invention finds a most efficient site on ovalbumin suitable for the CRISPR/Cas9 to cut and the vector to knock in the site. The site is positioned on a second exon of an ovalbumin gene, and the sequence of the site is shown in a sequence table No. 4. The procedure for determining this site is as follows: firstly, carrying out predictive analysis on a target area through online software (https:// www.benchling.com/criprpr /), and preliminarily determining 4 alternative sites; and then, carrying out in-vitro culture on chicken embryo fibroblasts, transfecting by using the CRISPR/Cas9 transient expression vector targeting the 4 sites, and carrying out CRISPR/Cas9 gene editing efficiency detection by using a T7E1 enzyme cutting method, wherein the highest detection positive rate is the final knock-in site.

As a further optimization of the present invention, an adenovirus vector titer detection step can be added between the above step (1) and step (2). The process is summarized as follows: culturing human kidney epithelial cell line (293T cell) in vitro, and after the growth curve enters an exponential phase, plating in a 48-hole culture dish; when the confluence degree of the plated 293T cells 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 vectors is judged according to the plaque condition on the cells, and the titer of the adenovirus vectors is calculated according to the infection rate and the dilution factor. Only if the titer of the adenovirus vector is ensured to be not less than 1X 10¹⁰PFU/mL can be followed by subsequent chick embryo microinjection steps, otherwise it is difficult to obtain positive site-directed knock-in type gene-editing chickens.

As a further optimization of the present invention, if the final packaged adenovirus vector titer cannot reach 1X 10¹⁰PFU/mL, which is an improvement of step (2), is obtained by isolating primordial germ cells from the blood of 2.5 day chick embryos, infecting the separated primordial germ cells in vitro with an adenovirus vector, and injecting the adenovirus vector-infected primordial germ cells into new chick embryos. The improved scheme can improve the infection efficiency of the adenovirus vector to germ cells, but has the defect that the separation of the primordial germ cells in blood is a time-consuming and labor-consuming step, and the packaged virus titer can be ensured to reach 1 × 10¹⁰In the case of PFU/mL or more, microinjection of chick embryos is preferably performed using the original protocol.

The invention has the beneficial effects that:

the invention relates to a fixed-point knock-in type gene editing chicken, which optimizes a target protein gene and constructs a fixed-point knock-in carrier by using a gene engineering technology, utilizes the technology of a third generation gene editing tool at the forefront at present, namely CRISPR (Clustered regularly interspaced short palindromic repeats) to ensure that an exogenous gene is directionally and stably integrated into a chicken genome, and ensures that the exogenous protein is efficiently expressed and secreted into egg white; by breeding and expanding the gene editing chicken and collecting a large number of eggs, the target protein can be obtained from the egg white at low cost and high efficiency.

Compared with the prior recombinant protein expression system, the gene editing chicken bioreactor has the following advantages:

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

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

3. the egg white has simple components and low purification cost, and even does not need to be purified in certain applications;

4. the protein has no protease, contains antibacterial enzyme and has long stable 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 the contents of trastuzumab in egg white edited by different numbered genes.

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

FIG. 4 shows the inhibition assay of trastuzumab on breast cancer cell growth.

FIG. 5 is a graph of trastuzumab apoptosis assay for breast cancer cells.

Detailed Description

Example 1

Knock-in vector construction comprising trastuzumab sequences.

(1) Cloning of upstream and downstream homology arms: respectively synthesizing two pairs of primers, namely HR and HL, and additionally adding a 30bp sequence which is homologous with the inserted fragment to the 5' end of the downstream primer so as to facilitate the fusion of the DNA fragment in the next step; adding enzyme cutting sites BamHI and EcoRI at the 5' end of the upstream primer for enzyme cutting, and carrying out PCR amplification by taking the whole chicken blood genome as a template, wherein the amplification system is as follows:

the amplified product was subjected to electrophoresis using 2% agarose gel, followed by recovery and purification of a 500bp band in the gel using a gel recovery kit of Beijing Tiangen Biotech Co.

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

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

the amplified product was subjected to electrophoresis using 1.2% agarose gel, followed by recovery and purification of 1340bp band in the gel using gel recovery kit of Beijing Tiangen Biotech Co.

(4) Vector digestion and ligation: carrying out BamHI/EcoRI double digestion on the 1340bp fragment and the adenovirus vector, wherein the digestion system is as follows:

after digestion at 37 ℃ overnight, the mixture was purified using a DNA purification kit from Beijing Tiangen Biotech Ltd. 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 ℃ the reaction mixture was transformed into competent cells, which were plated on solid media containing ampicillin after transformation. And (3) coating a plate for 48 hours, selecting a single colony for amplification culture, and extracting the plasmid in the cultured bacterial solution by using a plasmid extraction kit of Beijing Tiangen Biotech limited.

(4) Identification of knock-in vectors: and (3) carrying out PCR amplification on the extracted plasmid by using primers HL-F and HR-R, wherein an amplification system is as shown in step (1), and after carrying out gel electrophoresis and gel recovery on an amplification product, handing over to Shanghai Biotech limited company for sequencing. If the sequencing result is in accordance with the expected design, the vector is successfully constructed, and the vector is stored at minus 20 ℃.

Example 2

Chick embryos were microinjected for 2.5 days.

The chick embryo microinjection procedure is shown in FIG. 1 and is described in detail below:

(1) purchasing hatching eggs which are newly laid within 12 hours, cleaning the hatching eggs by using 0.1 percent of benzalkonium bromide, drying the hatching eggs in the air, drawing a circle with the diameter of about 1.5 centimeters on the equatorial plane of the hatching eggs by using waterproof glue, and paying attention to check to ensure that the waterproof glue is sealed and does not leak water; the hatching eggs were incubated at 37.8 ℃ and 60% humidity with 90 egg transfer once per hour. Note that: if the eggs are not hatched temporarily, the eggs need to be stored in a freezer at the temperature of 13 ℃ to prevent the eggs from developing in advance; the waterproof glue is hardened for about 12 hours, the eggs are laid horizontally for hatching before hardening, and the eggs can be hatched with the big heads upwards after hardening.

(2) After hatching eggs were incubated for 2.5 days (55-60 hours, in which case the chick embryos developed for about the period HH 14-16), a hole having a diameter of about 7 mm was milled in the equatorial plane of the hatching egg at 14000 rpm using a spiral hole puncher. Note that: punching only grinds the eggshell calcification layer, and needs to ensure the eggshell membrane to be complete; in order to prevent the chick embryos from being damaged in the punching process, the chick embryos are punched from the side or from the lower side, and the chick embryos float right above the hatching eggs and are far away from the holes.

(3) A plurality of common eggs are taken, the eggshell membrane of the eggs is peeled off by an ophthalmological forceps, the eggs are cut into a square shape with the side length of about 1 cm by an ophthalmological scissors, and the eggs are soaked in Phosphate Buffer Solution (PBS) for standby. A few drops of PBS are dripped in the ring surrounded by the sealant to isolate the outside air from entering hatching eggs, then the eggshell membrane is scratched by a scalpel, the eggshell membrane is taken out by an ophthalmological forceps, and the chick embryos and the peripheral blood vessels of the chick embryos can be observed at the moment. Note that: all the instruments and reagents used were autoclaved; PBS dropping in the sealing rubber ring not only plays the function of isolating air and external pollutants, but also can enable the chick embryo to float upwards to be close to the punched hole, and simultaneously plays the effect of a magnifier, thereby facilitating the microinjection on the next step.

(4) The adenovirus vector was removed from the-80 ℃ freezer and slowly thawed in ice at zero degrees. 2 microliter is absorbed by a micro-injection needle, and reflux blood vessels in peripheral blood vessels of the chick embryos are searched under a body type microscope for micro-injection. And (4) after injection, covering the holes on the hatching eggs with the eggshell membrane in the step (3), and sucking the PBS in the sealing rubber ring by using absorbent paper, wherein the eggshell membrane for covering can be tightly attached to the punched holes, so that air leakage is avoided. And (4) sealing the upper parts of the small holes with new sealant, and placing the hatching eggs in an incubator to continue hatching.

Example 3

And (4) detecting the content of trastuzumab in the egg white.

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

a. coating of antibody, namely using PBS containing 5% of bovine serum albumin as a coating solution, diluting a mouse anti-human trastuzumab antibody (primary antibody) by 1:2000 times, adding the diluted antibody into an enzyme label plate according to 100 microliter of each well, and coating the antibody overnight at the temperature of 4 ℃.

b. Washing the plate, using a pipette to remove the coated primary antibody solution in the wells on the second day, adding about 300 μ l of washing solution per well (PBS solution containing 5% detergent Tween-20) for rinsing, and rinsing 4 times each for 5 minutes on a shaker at 100 rpm;

c. and (3) sealing, namely adding sealing liquid (5% bovine serum albumin PBS) into the ELISA plate according to 200 microliter per hole, coating the ELISA plate on a shaking table at 50 revolutions per minute at room temperature of 25 ℃ for 2 hours, and absorbing the sealing liquid in the hole by using a liquid transfer gun after coating.

d. And (3) diluting the sample and the standard solution, namely performing gradient dilution on the albumen to be detected containing the trastuzumab by using PBS (phosphate buffer solution), wherein each gradient dilution is 10 times, and the total number of the gradients is 12. Diluting 3 gradients by using common egg white diluent as negative control; commercial trastuzumab was used as a positive control, with 3 gradients equally diluted. Since egg white is more viscous, the initial dilution was 100-fold. The standard trastuzumab solution was diluted simultaneously with PBS for 10 gradients. The solutions were sequentially added to an ELISA plate at 100. mu.l per well. The samples and standards were incubated at 25 ℃ for 2 hours.

e. And (c) after the plate is washed according to the step (b), adding a goat anti-mouse antibody (enzyme-labeled secondary antibody) labeled by horseradish peroxidase and a labeled antibody into the enzyme-labeled plate according to the proportion of 1:5000 dilution per hole of 200 microliter. At each gradient. The solutions were sequentially added to an ELISA plate at 100. mu.l per well. The samples and standard were incubated at 25 ℃ for 1 hour.

f. After the plate is washed according to the step b, newly prepared developing solution and TMB substrate are added into the ELISA plate according to 200 microliter per hole, and the reaction is carried out for 15 minutes at room temperature and 25 ℃ in a dark place. Subsequently 50 microliters of stop solution was added to each well.

g. And (4) placing the ELISA plate into an ELISA reader, and detecting the absorbance value at 450 nm. According to the absorbance value of the standard, a standard curve was prepared by using Excel software. And taking the average value of the absorbance values of the egg white trastuzumab diluted according to each gradient as the final concentration value.

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

Example 4

Application of recombinant trastuzumab in inhibition of cancer cell growth

The biological activity of recombinant trastuzumab was determined in HER2 positive breast cancer cell lines BT-474 and ZR-75-1 cultured in vitro using commercial trastuzumab (from Herceptin, Roche, Switzerland) as an application control. The recombinant trastuzumab and the cancer cell lines are incubated, a fluorescence-labeled secondary antibody is added to be combined with the recombinant trastuzumab, the combination condition of the recombinant trastuzumab and HER2 protein on two breast cancer cell lines is judged by analyzing the average fluorescence intensity on the cell membranes through a flow cytometer, and the result is shown in figure 3, wherein in the two breast cancer cell lines, the combination strength of the recombinant trastuzumab prepared by the invention and the commercial trastuzumab has no significant difference in combination strength with the target protein within the concentration range of 0.01-10 mu g/mL.

The recombinant trastuzumab prepared by the method and the commercial trastuzumab are added into the culture media of two breast cancer cell lines, and the results show that: the recombinant trastuzumab prepared by the invention has no difference on cell growth inhibition with commercial trastuzumab in ZR-75-1 cells, the recombinant trastuzumab prepared by the invention has no difference on cell growth inhibition with commercial trastuzumab in BT-474 cells in groups of 0.1 mu g/mL, 0.5 mu g/mL and 1 mu g/mL, and the recombinant trastuzumab prepared by the invention has significantly higher cell growth inhibition effect than commercial trastuzumab in groups of 5 mu g/mL and 10 mu g/mL (figure 4). The flow cytometry analysis result after Annexin V staining shows that the recombinant trastuzumab prepared by the invention can more remarkably induce apoptosis in BT-474 cells at 1 mug/mL, 5 mug/mL and 10 mug/mL groups (figure 5).

Sequence listing

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Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

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

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Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

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Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg

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Glu Leu Pro Thr Phe Gly Gly Gly Thr Lys Val Val Ile Thr Val Ala

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

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

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

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

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

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His Ser Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro

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Gly Gly Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr

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Asp Tyr Tyr Met Ser Trp Val Arg Gln Pro Pro Gly Lys Ala Leu Glu

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Trp Leu Gly Phe Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr

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Ser Ala Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln

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

100 105 110

Thr Tyr Tyr Cys Ala Ser Leu Tyr Tyr Gly Tyr Gly Tyr Ala Met Asp

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Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ser Thr Lys Gly

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

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 a gene editing chicken bioreactor is characterized in that the nucleotide sequence is shown in a sequence table No. 1.

2. The two-stage protein encoded by the gene of claim 1, wherein the amino acid sequence is shown in sequence Nos. 2 and 3.

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

4. A CRISPR/Cas9 gene editing site suitable for the high-efficiency site-directed knock-in of the knock-in vector of claim 1, which is positioned on the second exon of the ovalbumin gene of chicken, and the sequence of the site is shown in the sequence table No. 4.

5. The method for expressing recombinant trastuzumab gene using a gene editing chicken bioreactor according to claim 1, comprising the steps of:

(1) packaging an adenovirus vector: packaging the site-specific knock-in vector and the vector containing the sequence of CRISPR/Cas9 gene editing tool into a high-infection-capacity adenovirus vector, wherein the titer of the adenovirus vector is not lower than 1 x 10¹⁰PFU/mL；

(3) and (3) detecting the concentration and the activity of a product: and (3) breeding and expanding a large number of the successfully knocked-in gene-edited chickens, reserving the cocks as breeding hens, collecting eggs of the hens, and separating egg white of the hens.

6. The method for expressing a recombinant trastuzumab gene using a gene editing chicken bioreactor according to claim 5, wherein the recombinant trastuzumab sequence knocked in the vector comprises a 2A linker peptide sequence and a chicken lytic enzyme signal peptide sequence.