阅读说明：本技术 一种鸡ace2真核表达重组质粒载体的构建、鉴定与表达方法 (Construction, identification and expression method of chicken ACE2 eukaryotic expression recombinant plasmid vector ) 是由 纪晓霞 张源淑 王换换 李帅 李志强 曹西月 于 2020-10-21 设计创作，主要内容包括：本发明公开了一种鸡ACE2真核表达重组质粒载体的构建、鉴定与表达方法,包括构建ACE2真核表达载体,转染CHO细胞,筛选稳定表达的pcDNA.3.1(+)-ACE2细胞株,获得具有生物学活性的ACE2蛋白并测定酶活。本发明具有：①快速：仅需要1小时②简单：无需酶切③高效：阳性率达95％以上④无缝：不引入额外序列等优点。ACE2具有抗炎、抗损伤、抗纤维化等机体保护功能,特别作为SARS-CoV和SARS-CoV-2主要功能性受体,具有保护肺损伤功能。目前缺乏生物活性的鸡ACE2蛋白,限制了其生物学功能的研究。本发明为深入研究鸡ACE2作为病毒受体在禽冠状病毒感染中发挥的生物学功能及其分子机制,开发以ACE2为冠状病毒靶向治疗药物奠定基础。所以本发明无论是从基础研究还是临床运用的意义都是十分重大的。(The invention discloses a construction, identification and expression method of chicken ACE2 eukaryotic expression recombinant plasmid vector, which comprises the steps of constructing ACE2 eukaryotic expression vector, transfecting CHO cells, screening pcDNA.3.1(+) -ACE2 cell strain with stable expression, obtaining ACE2 protein with biological activity and determining enzyme activity. The present invention has: the method is characterized by comprising the following steps: only 1 hour is needed, simple: does not need enzyme digestion and has high efficiency: the positive rate is more than 95 percent, and the method is seamless: no extra sequence is introduced, and the like. ACE2 has anti-inflammatory, anti-injury, and anti-fibrosis effects, and can be used as main functional receptor of SARS-CoV and SARS-CoV-2 for protecting lung injury. The lack of bioactivity of chicken ACE2 protein limits the research of its biological function. The invention lays a foundation for deeply researching the biological function and the molecular mechanism of the chicken ACE2 which is taken as a virus receptor and is exerted in the infection of the avian coronavirus and developing a medicine for targeted therapy of the coronavirus by taking the ACE2 as the virus receptor. Therefore, the significance of the invention is very important from both basic research and clinical application.)

1. A construction, identification and expression method of a chicken ACE2 eukaryotic expression recombinant plasmid vector is characterized by comprising the following steps:

step 1, chicken ACE2 gene PCR amplification and recovery: adopting chicken jejunum cDNA as a template, designing a PCR primer according to a primer design principle according to an NCBI chicken ACE2 sequence MK560199, and adding a pcDNA3.1(+) vector homology arm and an EcoRI enzyme cutting site before an upstream primer initiation codon ATG, wherein the primer sequence is shown as SEQ ID NO. 1;

step 2, the recombination of the vector and the target fragment adopts a homologous recombination mode for connection;

step 3, verifying the construction result of the pcDNA3.1(+) -ACE2 eukaryotic expression plasmid by colony PCR, enzyme digestion and sequencing;

step 4, transfecting the CHO cells by a liposome method, primarily screening by G418, and further screening by monoclone to ensure that the recombinant plasmid can be stably expressed in the CHO cells;

step 5, identifying the expression result of pcDNA3.1(+) -ACE2 recombinant by RT-PCR, Western-blot and immunofluorescence;

and 6, determining the enzyme activity of the ACE2 active protein by a double-antibody sandwich method.

2. The method for constructing and identifying the chicken ACE2 eukaryotic expression recombinant plasmid vector as claimed in claim 1, wherein in step 1, the reaction conditions of PCR amplification are as follows: pre-denaturation at 95 ℃ for 1min, denaturation at 95 ℃ for 20s, annealing at 59 ℃ for 20s, extension at 72 ℃ for 1min15s, complete extension at 72 ℃ for 5min, and 32 cycles.

3. The method for constructing and identifying the chicken ACE2 eukaryotic expression recombinant plasmid vector as claimed in claim 1, wherein in step 2, the reaction ligation system (20 uL): comprises 4uL pcDNA3.1(+) vector, 1uL target sequence, 1uL homologous recombinase, 4uL5 Xbuffer and 10uL triple distilled water in a 200uLEP tube, and the three elements are evenly mixed and connected for 10min at 50 ℃.

4. The method for constructing, identifying and expressing the chicken ACE2 eukaryotic expression recombinant plasmid vector as claimed in claim 1, wherein in step 4, the minimum lethal concentration of G418 is determined to be 800ug/mL after adding G418 for 14d of screening.

5. The method for constructing, identifying and expressing the chicken ACE2 eucaryon expression recombinant plasmid vector as claimed in claim 1, wherein in step 4, the CHO cells are divided into a blank group, a transfection blank plasmid pcDNA3.1 group and a transfection pcDNA3.1-ACE2 group, the transfection mixture is sucked and added into a 6-well plate, the blank group is not treated, the transfection mixture is discarded after culturing for 6h at 37 ℃ and 5% CO2, and PBS washing is carried out. Then, 2mL of DMEM/F12 (containing 10% FBS) was added thereto, and the culture was continued.

6. The method for constructing, identifying and expressing the chicken ACE2 eukaryotic expression recombinant plasmid vector as claimed in claim 1, wherein in step 5, in Western blot identification, the cell protein extraction step comprises washing 6-well plate for screening monoclonal cells twice with PBS, adding protein lysate, lysing for 15min, and scraping the cells into an EP tube with a cell scraper. Centrifuging at 4 deg.C and 12000g/min for 20min to obtain supernatant as the extracted protein solution. The BCA method measures protein concentration and unifies protein concentration to 4 mg/mL.

7. The method for constructing, identifying and expressing the chicken ACE2 eucaryon expression recombinant plasmid vector as claimed in claim 1, wherein in step 6, empty plasmid pcDNA3.1(+), pcDNA3.1(+) -ACE2CHO cell and blank control cell are transfected to extract cell protein, and ACE2 activity protease activity is determined by double antibody sandwich method.

Technical Field

The invention belongs to the technical field of biology, and relates to a method for constructing, identifying and obtaining a cell strain stably overexpressing ACE2 gene by using chicken ACE2(Angiotensin converting enzyme 2) eukaryotic expression recombinant plasmid vector.

Background

Angiotensin converting enzyme 2 (ACE 2) is a monocarboxypeptidase first reported in 2000, and as an important member of renin-Angiotensin system, plays an important role in regulating cardiovascular, renal and intestinal functions. In 2003, researches show that ACE2 is a main functional receptor of severe acute respiratory syndrome coronavirus (SARS-CoV), has the functions of mediating SARS-CoV invasion and cell fusion and the like, and researches prove that the expression level of ACE2 gene is closely related to the entering of SARS-CoV and the severity of diseases. The study in 2019 found that SARS-CoV-2 can enter cells using all ACE2 except mouse ACE2 as receptors, and invade infected cells with the cell receptor ACE2 as with SARS-CoV.

According to the research of virus source tracing, the coronavirus such as SARS-CoV and SARS-CoV-2 all uses animal as natural host or intermediate host, so the research of animal ACE2 receptor function helps to clarify the cross-species infection mechanism of the virus. However, the studies of ACE2 mainly focus on the biological functions of ACE2 in various mammals such as human, mouse, rat, zebrafish and the like, as well as in aquatic animals, including poultry, are not clear at present.

In the research of the invention, white feather broilers are used as research objects, the ACE2 gene is amplified from the jejunum tissue of the white feather broilers by applying RT-PCR and gene cloning technology, the gene is connected to a eukaryotic expression vector pcDNA.3.1(+) in a homologous recombination mode, a cell strain which stably expresses pcDNA.3.1(+) -ACE2 is obtained in CHO cells through G418 and monoclonal screening, so that the ACE2 protein with biological activity is obtained, and a foundation is laid for deeply researching the biological function and the molecular mechanism of the chicken ACE2 which is taken as a virus receptor and is exerted in the avian coronavirus infection and developing a coronavirus targeted therapy medicament by using the ACE 2.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a method for constructing, identifying and expressing a chicken ACE2 eukaryotic expression recombinant plasmid vector pcDNA3.1(+) -ACE2, and aims to provide a method for constructing, identifying and expressing a chicken ACE2 eukaryotic expression recombinant plasmid vector pcDNA3.1(+) -ACE2, which comprises the following steps: firstly, the gene ACE2 is used as a carrier to transfer a target gene ACE2 into a host cell; secondly, the ACE2 gene is largely replicated (called clone) in host cells, which lays a foundation for deeply researching the biological function and molecular mechanism of chicken ACE2 as a virus receptor in the avian coronavirus infection and developing a targeted therapeutic drug taking ACE2 as a coronavirus. The specific technical scheme is as follows:

a construction and identification method of chicken ACE2 eukaryotic expression recombinant plasmid vector comprises the following steps:

s1, 21-day-old white feather broilers, taking heart, liver, lung, kidney, ileum, caecum, jejunum and the like after neck exsanguination and sacrifice, rinsing with physiological saline water, and quickly freezing in liquid nitrogen. Extracting genome RNA, and reverse transcribing to cDNA. Designing an upstream primer and a downstream primer of ACE2 for ACE2 full-length gene amplification, respectively adding pcDNA3.1(+) vector homologous arms and EcoRI enzyme cutting sites on two sides of an ACE2 gene fragment, and amplifying by taking cDNA as a template to obtain the ACE2 gene full length. EcoRI is used for digesting the pcDNA3.1(+) vector and the ACE2 PCR product, the target fragment is recovered by cutting glue, and the pcDNA3.1(+) vector and ACE2 are connected in a homologous recombination mode. The plasmid pcDNA3.1(+) -ACE2 is obtained through bacterial liquid PCR, enzyme digestion and sequencing verification.

S2, setting the lethal concentration of G418 to be 0, 500ug/mL, 600ug/mL, 700ug/mL, 800ug/mL, 900ug/mL, 1000 ug/mL. CHO cells were seeded in 6-well plates at 30% density, 2mL DMEM/F12 medium was added to each well, G418 was added at different concentrations, and the mixture was mixed well. The cells were cultured for 14 days based on the total death of the cells at the lowest concentration.

S3, dividing the CHO cells into a blank group, a transfection empty plasmid pcDNA3.1(+) group and a transfection pcDNA3.1(+) -ACE2 group, sucking the transfection mixed solution, adding the transfection mixed solution into a 6-well plate, carrying out no treatment on the blank group, abandoning the transfection mixed solution after culturing for 6h, and washing with PBS. Then, 2mL of DMEM/F12 (containing 10% FBS) was added thereto, and the culture was continued.

S4, further screening a cell strain which stably over-expresses ACE2 protein by G418 primary screening and single cloning.

S5, RT-PCR, immunofluorescence and Western blot to identify the recombinant expression of pcDNA3.1(+) -ACE 2.

S6, extracting cell proteins from the cells transfected with the empty plasmids pcDNA3.1(+), pcDNA3.1(+) -ACE2CHO and the blank control cell respectively, and determining the activity of the ACE2 active protease by a double antibody sandwich method.

The invention has the beneficial effects that:

(1) according to the invention, a pcDNA3.1(+) homology arm and an EcoRI restriction enzyme cutting site are added in front of an ATG (initiation codon) of a PCR upstream primer, so that the obtained PCR product contains the homology arm of the pcDNA3.1(+), and can be recombined with the pcDNA3.1(+) vector DNA.

(2) The pcDNA3.1(+) vector selected by the invention comprises the following components: contains a high-efficiency enhancer simian vacuolating virus (SV40) and can promote the gene transfection activity; has human cytomegalovirus early Promoter (PCMV) and carries out high-water stable expression of target genes; contains T7 Multiple Cloning Site (MCS) to facilitate the insertion of foreign gene; neomycin (neo) phosphotransferase resistance genes, which facilitate the selection of stable cell lines, can be used to select target cells containing the vector.

(3) CHO cells, as the most commonly used mammalian cell expression host, have the following advantages: 1) has definite genetic background and stable physiological metabolic capability; 2) the genetic relationship is closest to human, and the foreign protein modification ability is accurate; 3) has perfect gene transfer and vector expression system; 4) the shear force is resisted, and the method is suitable for large-scale culture; 5) CHO cells produce more than 70% of recombinant proteins and are considered safe genetically engineered recipient cells by the FDA in the united states due to their immortality and many other advantages.

(4) The invention adopts a homologous recombination mode to recombine the vector and the target fragment, is not limited by the traditional cloning method, can keep excellent accuracy for various cloning applications no matter how the vector type or the composition of the insert fragment, and does not need the subcloning operation of the traditional connection method. Even if the size of the inserted fragment or the number of fragments is increased, the cloning success rate is still high and is faster than that of the traditional ligase method.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the identification result (2444bp) of ACE2 gene amplified by RT-PCR method in the present invention; wherein, M: DNA marker; lanes 1-7: obtaining an ACE2 fragment from the jejunum tissue of the white feather broiler through RT-PCR amplification;

FIG. 2 is a diagram showing the restriction enzyme digestion result of eukaryotic expression plasmid pcDNA3.1 (+); wherein, M: DL 5000; lanes 1-8: pcDNA3.1(+) plasmid

FIG. 3A is the result chart of the product of ACE2 gene amplified by PCR of bacterial liquid; wherein, M: DNA marker lane 1: negative control; lanes 2-7: pcDNA3.1(+) -ACE 2-Positive colonies 1-6;

FIG. 3B is the restriction enzyme analysis result of pcDNA3.1(+) -ACE2 recombinant plasmid; wherein, M: DNA marker; lane 1: pcDNA3.1(+) -ACE2 recombinant plasmid; lane 2: the pcDNA3.1(+) -ACE2 recombinant plasmid is subjected to single enzyme digestion;

FIG. 4 is a graph of the death of CHO cells at 6d of the G418 screen; wherein, con: blank control group; a to F: the final concentration of G418 is 500ug/mL, 600ug/mL, 700ug/mL, 800ug/mL, 900ug/mL, 1000 ug/mL;

FIG. 5 is a diagram showing the screening results of the CHO cell transfected by the pcDNA3.1(+) -ACE2 recombinant plasmid; wherein A: CHO cells before transfection; b: CHO cells 48h after transfection with pcDNA3.1(+) -ACE 2; c: after transfection of pcDNA3.1(+) -ACE2, CHO cells were selected 7d after G418 selection; d: after pcDNA3.1(+) -ACE2 transfection, CHO cells after 14d were selected by G418;

FIG. 6 is a diagram showing the results of RT-PCR to identify pcDNA3.1(+) -ACE2 recombinants; wherein, M: DL 5000; lane 1: blank control group; lane 2: pcDNA3.1(+) empty plasmid group; lane 3: pcDNA3.1(+) -ACE2 recombinant;

FIG. 7 is a diagram showing the results of immunofluorescence identifying recombinant expression of pcDNA3.1(+) -ACE 2;

FIG. 8 is a diagram showing the result of Western Blot identifying recombinant expression of pcDNA3.1(+) -ACE2 transfected for 48 h; wherein, lanes 1, 2: blank control group; lanes 3, 4: pcDNA3.1(+) empty plasmid group; lanes 5, 6: pcDNA3.1(+) -ACE 2;

FIG. 9 is a diagram showing the result of Western blot identification of recombinant expression of pcDNA3.1(+) -ACE2 from monoclonal screening; wherein, lanes 1-3: pcDNA3.1(+) empty plasmid group; lanes 4-12: pcDNA3.1(+) -ACE2 group;

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The present invention will be further described with reference to specific examples and comparative examples.

Example 1 acquisition of a fragment of ACE2 mesh

The primer design is designed according to a chicken ACE2 gene sequence (MK560199) in GenBank, the upstream primer and the downstream primer are respectively inserted into a pcDNA3.1(+) homology arm and an EcoRI enzyme cutting site, and the primers are synthesized by Nanjing engine science and technology Limited company. Reverse transcription of RNA into cDNA, PCR amplification with cDNA as template, reaction system: cDNA template 2uL, reaction conditions: 5min at 95 ℃; 20s at 95 ℃, 20s at 59 ℃, 1min at 72 ℃ for 15s, and 40 cycles; 10min at 72 ℃. The PCR amplification result is shown in FIG. 1, and a single band appears (see lanes 1-7), and the size of the band is between 2000 and 3000bp, which is consistent with the expected (2444 bp).

Example 2 construction and characterization of eukaryotic expression plasmid pcDNA3.1(+) -ACE2

The obtained EcoRI single-enzyme ACE2 PCR purified product was ligated with pcDNA3.1(+) plasmid and recovered. The ACE2 gene fragment was ligated to pcDNA3.1(+) eukaryotic expression vector by homologous recombination. Linker system (20 uL): comprises 4uL pcDNA3.1(+) vector, 1uL target sequence, 1uL homologous recombinase, 4uL5 Xbuffer and 10uL triple distilled water in a 200uLEP tube, and the three elements are evenly mixed and connected for 10min at 50 ℃. The ligation product is transformed into DH5 alpha competent cell, and the positive recombinant expression vector pcDNA3.1(+) -ACE2 is obtained by enzyme digestion identification of bacterial liquid PCR and EcoRI, and is sent to Shanghai Yingying biotechnology Limited company for sequencing, and the sequencing result is completely consistent with the sequence of MK 560199. The successful construction of pcDNA3.1(+) -ACE2 is demonstrated. The digestion result of the eukaryotic expression plasmid pcDNA3.1(+) is shown in FIG. 2, a single band (lanes 1-8) exists at 5438bp, and the size of the band is consistent with that of the plasmid pcDNA3.1(+) at 5438bp, which proves the successful digestion. The colony PCR verified result is shown in FIG. 3A, with a single band between 3000bp and 2000-. PCR identified the size of the inserted fragment of the vector consistent with that of the PCR amplified fragment. Indicating that the target fragment is inserted successfully in the positive colony. The results of the enzyme digestion verification are shown in FIG. 3B: after EcoRI single cleavage, 1 band of about 8000bp was found, which was consistent with the sum of pcDNA3.1(+) vector (5428bp) and the target gene (2444bp) (lane 2). This result confirmed the successful construction of the pcDNA3.1(+) -ACE2 recombinant vector.

The primer sequence (SEQ ID NO.1) is as follows:

ACE2-F：

ACE2-R：

sequencing the obtained gene fragment (SEQ ID NO. 2):

example 3 transfection and expression of eukaryotic expression plasmid pcDNA3.1(+) -ACE2

1. CHO cell culture

The culture medium is DMEM/F12 culture medium, and the culture conditions are 37 ℃ and 5% of an incubator.

2. Determination of the minimum lethal concentration of G418

1) The G418 lethal concentration is set to be 0, 500ug/mL, 600ug/mL, 700ug/mL, 800ug/mL, 900ug/mL, 1000 ug/mL.

2) The CHO cells are inoculated in a 6-well plate at a density of 30%, after the cells are attached to the wall, the solution is discarded, PBS is used for washing, 2mL of DMEM/F12 culture medium is added into each well, G418 with different concentrations is added, and the mixture is uniformly mixed.

3) The culture medium was changed once for 3 days, cultured for 10-14 days, and the cell state was observed every day based on the total cell death at the lowest concentration.

The death of CHO cells at 6d of the G418 screen is shown in FIG. 4. When the screening is carried out on the 6d day by G418, the CHO cell form of the blank control group is a fibroblast sample and grows vigorously; cells with added G418 were morphologically rounded and had varying degrees of death. CHO cell death worsened with increasing G418 concentration and days.

3. Liposome transfection CHO cell

The CHO cells were divided into a blank group, a transfected empty plasmid pcDNA3.1(+) group and a transfected pcDNA3.1(+) -ACE2 group, and the specific operation was performed according to the Lipofectamine 3000 kit instructions:

first, solution A and solution B in the transfection mixture are prepared. Then mixing the solution A and the solution B at a ratio of 1: 1, gently blowing and mixing uniformly, and incubating for 10min at 37 ℃. The transfection mixture was aspirated into a 6-well plate, the blank was not treated, incubated at 37 ℃ for 6 hours with 5% CO2, and then the transfection mixture was discarded and washed with PBS. Then, 2mL of DMEM/F12 (containing 10% FBS) was added thereto, and the culture was continued.

4. Stable expression of pcDNA3.1-ACE2 recombinant plasmid in CHO cell

1) G418 preliminary screening

When the cell growth density reaches 70% -80%, G418 is added for screening, and the final concentration reaches the lowest lethal concentration of the previous screening. The growth state of the cells was observed, and the G418 concentration was maintained after 48h of liquid change. The result of the transfection of the pcDNA3.1(+) -ACE2 recombinant plasmid into CHO cells and the addition of G418 for 14d is shown in FIG. 5, and the transfected plasmid group has cell survival, visible cell cluster distribution and resistant monoclone appearance. While the blank cells all died, indicating successful transfection.

2) Monoclonal screening of stable cell lines

Individual cell clusters were found by inverted microscope and marked on the bottom of the dish with a marker pen. The cloning loop was placed on top of the selected clone with sterile forceps and after confirmation under the microscope, cells in the cloning loop were digested with pancreatin (0.25%) and the digestion was stopped. And transferring the cell suspension, sufficiently blowing and beating the cell suspension to enable the cell suspension to exist singly, sucking 100uL to a 96-well plate, sufficiently blowing and beating the cell suspension uniformly, repeatedly diluting until each well only contains 1 cell, and culturing at 37 ℃ and 5% CO2 until the cells grow full. Then expanded into 6-well plates/dishes and finally tested for expression.

Example 4 identification of recombinant expression of pcDNA3.1(+) -ACE2

1. RT-PCR identification

Harvest about 2X 10⁷Transferring the cells/hole into an EP tube, adding Trizol to thoroughly homogenize, extracting total RNA of the cells according to the Trizol Reagent operation step, carrying out reverse transcription by a two-step method to obtain cDNA, carrying out PCR amplification by taking the cDNA as a template, and carrying out reaction conditions: 5min at 95 ℃; 20s at 95 ℃, 20s at 59 ℃, 1min at 72 ℃ for 15s, and 40 cycles;10min at 72 ℃. The PCR product was detected by electrophoresis on a 1% agarose gel. As a result, as shown in FIG. 6, the recombinant pcDNA3.1(+) -ACE2 showed a single band (lane 3), and the band size was between 2000 and 3000bp, which is consistent with the expected (2444bp) size. It was demonstrated that the pcDNA3.1(+) -ACE2 plasmid was successfully transfected into CHO cells.

2. Immunofluorescence assay

Cells after 48h of transfection screening were seeded on slides in 12-well plates at a cell density of 1X 10⁵Per well at 5% CO₂Culturing at 37 deg.C until the cell growth fusion degree is 80-90%, discarding culture solution, washing with PBS, fixing with 4% paraformaldehyde at room temperature for 10min, washing with PBS, adding 1% Triton X-100, standing at room temperature for 5min to make cell membrane permeable. Discarding the supernatant, washing with PBS, blocking with 5% BSA at room temperature, adding home-made chicken ACE2 polyclonal antibody diluted at a ratio of 1: 400, and incubating overnight at 4 ℃; after washing, adding FITC labeled goat anti-mouse IgG (diluted 1: 1000), and incubating at room temperature in a dark place; DAPI staining solution was added, light-protected staining was performed, and PBS washing was performed. The cell slide was gently placed on a slide and mounted with an anti-fluorescence decay mounting agent. Cells were scanned and observed under an LSM 510 laser confocal microscope. DAPI excitation light wavelength 358nm, emission light wavelength 461 nm; the FITC excitation wavelength is 495nm, and the emission wavelength is 520-530 nm. As shown in FIG. 7, pcDNA3.1(+) -ACE2 was expressed in CHO cells and was distributed evenly over the cell membrane. The fluorescence intensity (green fluorescence) of the pcDNA3.1(+) -ACE2 recombinant plasmid group is obviously higher than that of a blank control group and that of the pcDNA3.1(+) empty plasmid group, and the result shows that the pcDNA3.1(+) -ACE2 recombinant plasmid is successfully transfected into a CHO cell and has higher transfection efficiency.

3. Western Blot identification

Extracting cell protein: the 6-well plate from which the monoclonal cells were selected was washed twice with PBS, added with protein lysate, lysed for 15min, and the cells were scraped into an EP tube with a cell scraper. Centrifuging at 4 deg.C and 12000g/min for 20min to obtain supernatant as the extracted protein solution. The BCA method measures protein concentration and unifies protein concentration to 4 mg/mL. After SDS-PAGE electrophoresis, cell proteins are electrically transferred to a PVDF membrane under the condition of constant current of 100V for 90 min. Blocking with 5% skimmed milk at room temperature for 2h, washing with PBST for 3 times; using mouse anti-chicken ACE2 polyclonal antibody (1: 3000) as primary antibody, incubating at room temperature for 2h, and washing with PBST for three times, each time for 10 min; horseradish peroxidase-labeled goat anti-mouse IgG is used as a secondary antibody (1: 5000), incubated for 1h at normal temperature, washed with PBST for three times, each time for 10min, and finally exposed to ECL luminescence solution. Transfection for 48h, Western Blot to identify transfection efficiency results are shown in FIG. 8, showing significant bands at around 105kDa (lanes 5, 6), consistent with the expected size of ACE 2; the blank control and the negative control had a relatively shallow ACE2 band (lanes 1-4), and the expression level was significantly lower. It is demonstrated that pcDNA3.1(+) -ACE2 plasmid has been successfully transfected into CHO cell, and ACE2 gene has been integrated into cell chromosome, and ACE2 protein is expressed. After selecting the monoclone, extracting protein from the CHO cell stably transfected with the pcDNA3.1(+) -ACE2 plasmid, detecting by Western Blot, finding that 4 strains of cells have significant bands (lanes 4, 5, 7 and 8) at nearly 105kDa as shown in figure 9, and respectively naming the 4 strains of cells as pcDNA3.1(+) -ACE2-A, pcDNA3.1(+) -ACE2-B, pcDNA3.1(+) -ACE2-C and pcDNA3.1(+) -ACE 2-D.

Example 5 ACE2 Activity protease Activity assay

Cell proteins are extracted from the cells transfected with the empty plasmids pcDNA3.1(+), pcDNA3.1(+) -ACE2CHO and the blank control cells respectively, and the activity of the ACE2 active protease is determined by a double-antibody sandwich method. The specific operation is carried out according to the kit instructions. And (3) adjusting the blank hole to zero, measuring the absorbance value of each hole at the wavelength of 450nm, obtaining a standard curve and a regression equation according to the standard substance, and calculating the enzyme activity of the ACE2 active protein. Unit: U/L. The two-antibody sandwich method is used for measuring the activity of the ACE2 active protease. The result is shown in Table 1, the enzyme activity of the ACE2 active protein of the cell strain pcDNA3.1(+) -ACE2-A reaches 601.77U/L, which is remarkably higher than that of a blank control group, and the result shows that the obtained ACE2 active protein has higher enzyme activity and can be used for subsequent research.

TABLE 1 ACE2 enzyme Activity assay results

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.