CN113122523A - Cloning of chicken ACE2 gene, expression and purification of protein and preparation of polyclonal antibody thereof - Google Patents
Cloning of chicken ACE2 gene, expression and purification of protein and preparation of polyclonal antibody thereof Download PDFInfo
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Abstract
The invention provides cloning of chicken ACE2 gene, expression and purification of protein and preparation of polyclonal antibody thereof. Provides an amino acid sequence and a gene sequence of the ACE2 protein, constructs an ACE2 prokaryotic expression vector, expresses the ACE2 protein by using an escherichia coli prokaryotic expression system, and purifies the protein. The purified protein can be used for preparing polyclonal antibodies with higher titer, and the antigen and the polyclonal antibodies in the invention can lay a foundation for further development of detection kits. ACE2 is used as negative regulation molecule of RAS system, and has effects of resisting injury, resisting fibrosis, etc. by targeting degradation of AngII; as a main functional receptor of acute severe respiratory syndrome coronavirus (SARS-CoV), has the function of protecting lung injury; ACE2 has an important effect on the poor absorption of amino acids, the intestinal microecological balance and the gastrointestinal inflammation in cooperation with the transportation of amino acid transport carriers. Therefore, the present invention is very important in both basic research and clinical application.
Description
Technical Field
The invention belongs to the technical field of molecular cloning, and particularly relates to cloning of a new chicken gene ACE2 (Angiotensin converting enzyme 2), expression and purification of protein and preparation of a polyclonal antibody.
Background
Angiotensin converting enzyme 2(ACE2) is a member of the ACE homologs discovered in 2000 and is an important regulator of the renin-angiotensin system. Belongs to type I transmembrane glycoprotein, and is a zinc ion-containing monocarboxypeptide glycoprotein. It is widely found in the heart, liver, lung, kidney, testis, and intestinal tract of animals. ACE2 degrades angiotensin II (AngII) into angiotensin (1-7) [ Ang (1-7) ] with vasodilating and antiproliferative effects, and also degrades angiotensin I (AngI) into Ang (1-9). Ang (1-7) plays roles of anti-oxidation, anti-fibrosis, anti-inflammation and the like through a receptor Mas (MasR), and attracts extensive attention of researchers.
Research on ACE2 is currently focused on the following three areas: ACE2 is used as negative regulation molecule of RAS system, and has effects of resisting injury, resisting fibrosis, etc. by targeting degrading AngII; the compound is used as a main functional receptor of acute severe respiratory syndrome coronavirus (SARS-CoV) and has the function of protecting lung injury; ACE2 has an important effect on the poor absorption of amino acids, the intestinal microecological balance and the gastrointestinal inflammation in cooperation with the transportation of amino acid transport carriers.
Human, mouse, rat, cat, sheep, pig, zebra fish and other mammals and aquatic animals, and the ACE2 gene of mammals is identified and confirmed. However, in birds, the ACE2 gene has been recently reported, and particularly the ACE2 gene in poultry chickens has not been annotated.
In the research of the invention, according to the predicted chicken ACE2 sequence published by NCBI, the ACE2 gene of chicken is obtained by PCR amplification for the first time, and pET32a-ACE2 are expressed by utilizing a prokaryotic expression system
Recombinant protein, and preparing the mouse anti-ACE 2 serum polyclonal antibody by purifying pET32a-ACE2 recombinant protein. The research of the invention fills the blank of the lack of the full-length sequence of the chicken ACE2 in GenBank, and provides basic data for the research of ACE2 in broiler chicken and poultry.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to play the body protection functions of resisting injury, resisting fibrosis and the like by targeting degradation of AngII according to the fact that ACE2 is used as a negative regulation molecule of an RAS system; as a main functional receptor of acute severe respiratory syndrome coronavirus (SARS-CoV), has the function of protecting lung injury; the method has the advantages that the transfer of the amino acid transport carrier is coordinated, the key effects of the transfer of the amino acid transport carrier, the amino acid malabsorption, the intestinal microecological balance and the gastrointestinal inflammation are exerted, the ACE2 gene of the chicken is not annotated, the full length of the ACE2 gene of the chicken is obtained by cloning, the blank of the lack of the full length sequence of the ACE2 gene in GeneBank is filled, the ACE2 protein is expressed by pronucleus, the corresponding polyclonal antibody is prepared, and basic data can be provided for the research of the ACE2 on broiler chickens and poultry.
In order to achieve the purpose, the invention adopts the following technical scheme that:
an ACE2 protein of chicken, the amino acid sequence of which is shown in SEQ ID NO. 1.
A gene for coding the ACE2 protein of the chicken, wherein the gene codes an amino acid sequence shown as SEQ ID NO.1, or a nucleotide sequence of the gene is shown as SEQ ID NO. 2.
A connection vector or an expression vector of chicken ACE2, wherein the connection vector or the expression vector contains a nucleotide sequence shown as SEQ ID NO. 2.
A method for preparing chicken ACE2 cloning vector or expression vector, which 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. Genomic RNA was extracted and the RNA was reverse transcribed into cDNA using random primers. Designing an upstream primer and a downstream primer of ACE2 for ACE2 full-length gene amplification, respectively adding BamHI and HindIII enzyme cutting sites on two sides of an ACE2 gene fragment, and obtaining the ACE2 gene full length by amplification by taking cDNA as a template, wherein the amplification conditions are as follows: 5min at 95 ℃; 20s at 95 ℃, 20s at 59 ℃, 1min at 72 ℃ for 15s, and 40 cycles; 10min at 72 ℃.
S2, cutting gel to purify a target fragment, taking 0.5uL pMD19-T vector, 4.5uL purified product and 5uLSolution I in a 200uLEP tube, mixing uniformly, running on a PCR instrument for 16 ℃, and connecting for 14 h. And carrying out PCR (polymerase chain reaction), enzyme digestion and sequencing verification on a bacterial liquid to obtain a plasmid pMD19T-ACE 2.
The preparation method as described above preferably further comprises digesting pET32a vector and pMD19T-ACE2 plasmid with BamHI and HindIII respectively, cutting the gel to recover the target fragment, ligating the recovered pET32a vector with recovered pMD19T-ACE2 recombinant plasmid with T4 ligase, and ligating the resultant system (10 uL); 1uL pET32a vector, 7uL target sequence, 1uLT4 ligase, 1uL10 xbuffer are put in a 200uLEP tube, mixed evenly, operated on a PCR instrument at 22 ℃ for 14h for connection, and the plasmid pET32a-ACE2 is obtained through bacterial fluid PCR, enzyme digestion and sequencing verification.
A method for preparing chicken ACE2 protein comprises transferring chicken ACE2 prokaryotic expression vector into DH5 alpha and BL21 competent cells, and performing IPTG induced expression to obtain pET32a-ACE2 recombinant protein.
The preparation method comprises the steps of transferring the plasmid pET-32a-ACE2 into DH5 alpha and BL21 competent cells, inducing and expressing pET-32a-ACE2 protein to exist in the form of inclusion bodies, and purifying: preparing 10% separation gel and 5% concentrated gel, without inserting comb, adding 500uL of inclusion body extract treated by sample buffer after gel solidification, preparing gel by conventional method, adding standard molecular weight protein as reference, and performing SDS-PAGE by conventional method.
After electrophoresis, putting the discharged gel into a clean large plate, dyeing for 10min by 0.25mol/LKCl on a shaking table, cutting off a silver-white target band by using an operating blade after dyeing, washing for 5 times by using distilled water, transferring the gel into a clean small bag, adding 500uLPBS, shaking and uniformly mixing, repeatedly freezing and thawing for 3 times at-20 ℃, 12000r/min, centrifuging for 3min, and taking supernatant SDS-PAGE electrophoresis and Western blot for detecting and purifying effects.
The preparation of chicken ACE2 protein polyclonal antibody comprises the following steps: the obtained chicken pET32a-ACE2 recombinant protein immune Wistar rat is inoculated in a mode of subcutaneous multi-point injection at the neck and the back, a Freund complete adjuvant is used as a primary immune, a Freund incomplete adjuvant is used as a secondary immune, a tertiary immune and a quarternary immune respectively, and rat serum is taken three days after the boosting immunity.
The invention has the beneficial effects that:
the invention firstly amplifies the ACE2 gene of chicken, not only fills the blank of the lack of the full-length sequence of the ACE2 in the GenBank, but also provides basic data for the research of ACE2 in the aspects of broilers and poultry.
The invention adopts an escherichia coli prokaryotic expression system, constructs pET32a-ACE2 plasmid vector, and performs protein expression and purification, thereby not only being capable of preparing polyclonal antibody with higher titer, but also laying a foundation for the research of related functions of ACE 2.
The amplified ACE2 gene is obtained by the invention, and the function research of the ACE2 is only focused on the following three aspects: ACE2 is used as negative regulation molecule of RAS system, and has effects of resisting injury, resisting fibrosis, etc. by targeting degrading AngII; the compound has the function of protecting lung injury as a main functional receptor of acute severe respiratory syndrome coronavirus (SARS-CoV); ACE2 has an important effect on the poor absorption of amino acids, the intestinal microecological balance and the gastrointestinal inflammation in cooperation with the transportation of amino acid transport carriers. ACE2 may provide a new idea for treating intestinal inflammation of chicken, and lays a foundation for sustainable development of poultry industry.
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 embodiments or the prior art descriptions will be briefly introduced 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 1-4: obtaining an ACE2 fragment from the jejunum tissue of the white feather broiler through RT-PCR amplification; lane 5: negative control; lane 6: 18SrRNA gene;
FIG. 2A is the result chart of the product of ACE2 gene amplified by PCR of bacterial liquid; wherein, M: DNA marker: lanes 1, 2: pMD-19T-ACE 2-Positive colony lane 3: negative control;
FIG. 2B is a single and double restriction enzyme identification result diagram of pMD-19T-ACE2 recombinant plasmid; wherein, M: DNA marker; lanes 1, 4: pMD-19T-ACE2 recombinant plasmid 1; lanes 2, 5: pMD-19T-ACE2 recombinant plasmid 1 is subjected to single enzyme digestion; lanes 3, 6: pMD-19T-ACE2 recombinant plasmid 1 is subjected to double enzyme digestion;
FIG. 3A is a diagram showing the double digestion result of pET32a plasmid, wherein, M: DL 10000; lanes 1-8: the pET32a plasmid;
FIG. 3B is a diagram of the double cleavage result of pMD-19T-ACE2 recombinant plasmid, wherein M: DL 10000; lanes 1-7: pMD-19T-ACE2 recombinant plasmid;
FIG. 4A is a graph of monoclonal colony results for DH5 alpha competent cells;
fig. 4B is a result diagram of PCR amplification products of ACE2 gene bacterial liquid, wherein, M: DL 10000; lane 1: pET32a-ACE 2-positive colony 1; lane 2: pET32a-ACE 2-positive colony 2; a swimming channel 3: pET32a-ACE 2-positive colony 3; lane 4: pET32a-ACE 2-positive colony 4; lane 5: pET32a-ACE 2-positive colony 5; lane 6: pET32a-ACE 2-positive colony 6;
FIG. 4C is a single-and double-restriction enzyme identification result chart of pET32a-ACE2 recombinant plasmid, wherein M: DL 10000; 1: pET32a-ACE2 recombinant plasmid 1; 2: single enzyme digestion of pET32a-ACE2 recombinant plasmid 1; 3: double digestion of pET32a-ACE2 recombinant plasmid 1; 4: pET32a-ACE2 recombinant plasmid 2; 5: the pET32a-ACE2 recombinant plasmid 2 is subjected to single enzyme digestion; 6: double digestion of pET32a-ACE2 recombinant plasmid 2;
fig. 5A is a graph showing the results of IPTG stimulation of pET32a-ACE2 recombinant protein expression at different concentrations, where M: molecular mass of standard protein; lanes 1-8: the expression condition of pET32a-ACE2 recombinant protein induced by IPTG with final concentration of 0, 0.125, 0.25, 0.5, 1, 2, 4 and 8 mmol/L;
FIG. 5B is a graph showing the results of IPTG-induced expression of pET32a-ACE2 recombinant protein at different times, wherein M: molecular mass of standard protein; lanes 1-9: respectively inducing the expression conditions of the pET32a-ACE2 recombinant proteins for 0h, 2h, 4h, 6h, 8h, 10h and 12h, staying overnight and staying overnight by 1mmol/L IPTG;
FIG. 6 is a graph showing the results of identifying the expression form of pET32a-ACE2 recombinant protein, wherein M: the molecular mass of the standard protein; lanes 1, 5: after induction, expressing a product by pET32a-ACE2 recombinant bacteria; lanes 2, 6: ultrasonically crushing an expression product of the pET32a-ACE2 recombinant bacteria after induction; lanes 3, 7: ultrasonically crushing a supernatant of the pET32a-ACE2 recombinant bacteria after induction; lanes 4, 8: ultrasonically crushing and precipitating pET32a-ACE2 recombinant bacteria after induction;
FIG. 7A is a SDS-PAGE result of KCL gel-cut purified pET32a-ACE2 inclusion body protein, wherein, M: molecular mass of standard protein; lane 1: after induction, expressing a product by pET32a-ACE2 recombinant bacteria; lane 2: ultrasonically crushing an expression product of the pET32a-ACE2 recombinant bacteria after induction; lane 3: KCL gel cutting and purifying pET32a-ACE2 recombinant protein; lane 4: repeatedly freezing and thawing the KCL gel-cutting purified pET32a-ACE2 recombinant protein for 3 times;
FIG. 7B is a WB validation result of KCL gel-cut purified pET32a-ACE2 inclusion body protein, wherein, lane 1: unpurified pET32a-ACE2 inclusion body protein; lane 2: KCL gel cutting purification pET32a-ACE2 inclusion body protein;
FIG. 8 is a graph showing the results of the potency assay for the murine anti-chicken ACE2 polyclonal antibody;
fig. 9 is a graph showing the results of immunoblotting for detecting serum polyclonal antibodies, wherein a: positive serum 1-3: pET32a-ACE2 recombinant protein; b: negative sera 1-3: pET32a-ACE2 recombinant protein;
FIG. 10 is a graph showing the results of detecting ACE2 protein expression in chicken tissues by using the antibody of the present invention, wherein a lane 1: a heart; lane 2: a liver; lane 3: the lung; lane 4: the kidney; lane 5: jejunum; and (6) swimming channel: the ileum; lane 7: the duodenum; lane 8: the cecum; lane 9: a rectum;
FIG. 11 is a graph showing the results of detecting ACE2 protein expression in different tissues of the antibody of the present invention, wherein lane 1: chicken ileum; lane 2: chicken duodenum; lane 3: sheep ileum; lane 4: sheep duodenum; lane 5: rat ileum; lane 6: rat duodenum; lane 7: the porcine ileum; lane 8: a porcine duodenum;
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 obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as is well understood by those of skill 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 amplification of ACE2 Gene and construction of ligation vector
The primer design is designed according to the predicted chicken ACE2 gene sequence (XM-416822.5) in GenBank, and BamHI and HindIII enzyme cutting sites are respectively inserted into upstream and downstream primers to synthesize the primers by Nanjing Optimak Biotechnology Limited. 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 appeared (see lanes 1-4), the band size was between 2000 and 3000bp, which is consistent with the expected (2444bp) size.
And purifying and recovering the PCR product by using a DNA gel recovery kit, and performing the specific steps according to the instruction of the DNA gel recovery kit. The recovered product was ligated with the pMD19T vector (16 ℃, 14h), and the ligation product was transformed into competent cells DH5 α. The cells were resuspended and spread on LB/Amp/X-Gal/IPTG medium plates and cultured at 37 ℃ for 16 hours. The single colonies on the petri dish were inoculated in a liquid medium containing ampicillin and cultured overnight. Plasmid is extracted by a small plasmid extraction kit, the extracted plasmid is verified by HindIII single enzyme digestion and BamHI and HindIII double enzyme digestion (37 ℃, 14h), and the identified positive plasmid is sent to Shanghai Yingjun biotechnology company Limited for sequencing. The colony PCR verified result is shown in FIG. 2A, 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 successful insertion of the target fragment in the positive colonies. The results of the enzyme digestion verification are shown in FIG. 2B: after single digestion, Hind III found about 1 band of 5136bp (lanes 2 and 5), which was consistent with the sum of pMD-19T vector (2692bp) and target gene (2444 bp). The double digestion with BamHI and Hind III revealed two bands of 2444bp and 2692bp (lanes 3 and 6). This result confirmed the successful construction of the pMD-19T-ACE2 recombinant vector. Sequencing the obtained gene fragment (SEQ ID NO. 2):
the coding amino acid sequence is as follows (SEQ ID NO. 1):
the primer sequences are as follows:
ACE2-F(SEQ ID NO.3)：CGGGATCCATGTTGCTTCACTTCTGGCTTCTCTGTG
ACE2-R(SEQ ID NO.4)：GCGAAGCTTCTAAAAGGATGTTTGTGTCTCTTCAGATTGCT CA
EXAMPLE 2 protein expression experiment
The correct pMD19T-ACE2 recombinant plasmid and pET32a prokaryotic expression vector are identified by utilizing HindIII/BamHI double enzyme digestion through sequencing, a pET32a plasmid double enzyme digestion result diagram is shown in a figure 3A, the pET32a plasmid is verified by BamHI and HindIII double enzyme digestion, enzyme digestion products are identified by 1% agarose gel electrophoresis, a single target band is found at 5900bp, the other band is about 50bp, and the diagram is not shown. The enzyme digestion is proved to be successful. The result of double digestion of the pMD-19T-ACE2 recombinant plasmid is shown in FIG. 3B, and double digestion of the pMD-19T-ACE2 recombinant plasmid BamHI and HindIII can show two bands of 2444bp and 2692bp, which proves successful digestion.
The ACE2 gene fragment is recovered and connected to pET32a prokaryotic expression vector, the result of the single colony of DH5 alpha competent cell and DH5 alpha competent cell is shown in figure 4A, and the white spot shows that the recombinant plasmid is successfully transferred to form the single colony. The PCR amplification product result of the bacterial liquid PCR and HindIII/BamHI double enzyme digestion identification is shown in FIG. 4B, wherein the positive recombinant expression vector pET32a-ACE2 is obtained, the PCR amplification product result of the ACE2 gene bacterial liquid PCR is shown in lanes 1 and 2, and the size of the band is consistent with that of the expected band (2444 bp). The single and double restriction identification results of pET32a-ACE2 recombinant plasmid are shown in FIG. 4C, 1 band of about 8344bp is found after single restriction of HindIII, and the size of the band is consistent with the sum of pET32a vector (5900bp) and target gene (2444bp) (lanes 2 and 5). Two bands of about 5900bp and 2444bp were observed with BamHI and HindIII (lanes 3 and 6). This result confirmed the successful construction of pET32a-ACE2 recombinant vector. The plasmid was sent to Shanghai Junjun Biotechnology Co., Ltd for sequencing.
Converting a pET32a-ACE2 recombinant plasmid with correct sequencing identification into BL21(DE3) competent cells, selecting a single colony, selecting a white colony to be inoculated in a 20mLLB culture medium, performing shake culture at 37 ℃ until the OD600 is 0.6-0.8, taking 1mL of bacterial liquid as a control before induction, and taking 1mL of residual bacterial liquid to respectively add 20mg/mLIPTG to the final concentration of 8, 4, 2, 1, 0.5, 0.25 and 0.125 mmol/L. After 6h of induction at 28 ℃, collecting 1mL of bacterial liquid, centrifuging at 8000rpm/min for 15min, collecting bacterial liquid precipitate, resuspending the precipitate at 100uLPBS, and determining the optimal induction concentration of IPTG through SDS-PAGE electrophoresis; picking white colony to add 20mLLB culture medium, shake culturing at 37 deg.C until OD600 is 0.6-0.8, taking 1mL bacterial liquid as control before induction, taking 1mL residual bacterial liquid, and adding 20mg/mLIPTG to final concentration of 1 mmol/L. Inducing at 28 deg.C for 2, 4, 6, 8, 10h, collecting 1mL bacterial liquid, centrifuging at 8000rpm/min for 15min, collecting bacterial liquid precipitate, resuspending the precipitate at 100uLPBS, and determining IPTG optimal induction time by SDS-PAGE electrophoresis. As shown in FIG. 5, the optimization result of pET32a-ACE2 recombinant protein induction conditions shows that IPTG can really induce the expression of the target protein, and the target protein expression level is higher when the final concentration of IPTG is 0.5, 1, 2 and 4mmol/L, so that the induction concentration of the screened IPTG is 1 mmol/L. The expression level of the recombinant protein increased with the increase of the induction time, but the expression level of the recombinant protein did not increase significantly after 8 hours of induction and further increase of the induction time, and therefore the induction time was determined to be 10 hours.
Selecting IPTG induction concentration and time with high expression quantity, carrying out mass induction expression on pET32a-ACE2 recombinant protein, collecting bacterial liquid after inducing for 10h at 28 ℃, centrifuging for 15min at 8000rpm/min, collecting bacterial liquid precipitate, washing for 2 times by PBS, cracking thalli by ultrasonic waves, cracking for 5s by ultrasonic waves, separating for 10s, and analyzing SDS-PAGE to identify the expression form of the target protein. As shown in FIG. 6, the content of the target protein in lanes 4 and 8 (precipitates) is significantly higher than that in lanes 3 and 7 (supernatants), i.e., the pET32a-ACE2 recombinant protein exists mainly in the form of inclusion bodies.
Example 3 protein purification experiment
Preparing 10% separation gel and 5% concentration gel, without inserting comb, adding 500uL of inclusion body extract processed by loading buffer solution after the gel is solidified, preparing gel by conventional method, adding standard molecular weight protein as reference, and performing SDS-PAGE by conventional method.
After electrophoresis, the discharged gel is put into a clean big plate, 0.25mol/LKCl is dyed for 10min on a shaking table, after dyeing, a silver-white target band is cut off by a surgical blade, after 5 times of washing by distilled water, the gel is moved into a clean small bag, 500uLPBS is added to shake and mix evenly, freezing and thawing are carried out repeatedly for 3 times at the temperature of-20 ℃, 12000r/min is carried out, and the supernatant SDS-PAGE electrophoresis and Westernblot are taken out after 3min of centrifugation for purity testing. Westernblot identification of ACE2 recombinant protein comprises the following specific operations: after SDS-PAGE electrophoresis is carried out on the purified ACE2 recombinant protein and pET32a vector induced expression bacteria, a corresponding band on the gel is transferred to a PVDF membrane by a wet transfer method under the constant current of 100V for 90 min. ACE2 primary antibody diluted 1: 3000, horseradish enzyme labeled goat anti-rabbit IgG diluted 1: 5000. The identification result of the gel-cut purified recombinant protein is shown in fig. 7, a single-mesh band (100kDa) is observed compared with the non-purified group, and the content of the obtained purified protein is higher.
Example 4 preparation of ACE2 polyclonal antibody
1. Immunization of Wistar rats.
4 SPF-grade Wistar female rats (7 weeks old) were purchased from the laboratory animal center of Yangzhou university (laboratory animal permit: SCXK (su) 2017-. Rats, randomly divided into 1 control group and 3 experimental groups, were weighed, separately, blood was collected by tail-off (as negative control), and then immunized according to the following procedure.
First immunization: 600uL of Freund's complete adjuvant and 600uL of prepared antigen (purified recombinant protein of white feather broiler pET32a-ACE2, concentration is 1402ug/mL) were mixed well to complete emulsification. The emulsified antigen liquid is injected subcutaneously into mice, and the experimental group is injected subcutaneously into dorsoventral part at multiple points, wherein each part is 300 uL. The control group was also injected subcutaneously in multiple dorsal abdominal sites with 1.2mL of Freund's complete adjuvant. The second, third and fourth immunizations used Freund's incomplete adjuvant, and the immunization period was 28 days for 4 times. On the 7 th day after 4 times of immunization, blood is collected from the orbit of the rat, the blood is collected and inactivated at 37 ℃ for 2 hours, the blood is coagulated overnight at 4 ℃ to release serum, and the coagulated blood is 10000r/min and 10 min; and collecting the supernatant to obtain the serum.
2. ELISA for determining serum titer
The serum titer was determined using an indirect ELISA method. The serum titer of rats was determined by using purified pET32a-ACE2 protein as the coating antigen at a concentration of 403ug/mL overnight at 4 ℃. As shown in FIG. 8, the serum titer of 6 mice was 1: 6400, compared to the negative control.
EXAMPLE 5 identification of polyclonal antibodies
1. Western Blot identification of purified recombinant protein
And (3) carrying out SDS-PAGE gel electrophoresis on the purified ACE2 recombinant protein, and then electrically transferring 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; rat positive (1: 5000), negative (1: 5000) and ACE2 monoclonal antibody (1: 3000) were used as primary antibodies, incubated at room temperature for 2h, washed three times with PBST, 10min each; 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.
The results are shown in FIG. 9, which shows that the immunized rat serum can specifically bind to ACE2 protein (A), and the negative mouse serum cannot bind to ACE2 protein (B).
2. Western Blot identification of ACE2 protein in tissues
(1) Western Blot identification of ACE2 protein in various tissues of white feather broiler chicken
Extracting the white feather broiler heart, liver, lung, kidney, ileum, caecum and jejunum tissue proteins by adopting a two-step method, determining the concentration of each tissue protein by using a BCA kit, diluting each tissue protein to the same concentration, and storing at-20 ℃.
And (3) performing SDS-PAGE electrophoresis on the total tissue protein extracted in the previous step, concentrating the gel by 5 percent, and separating the gel by 10 percent. The working voltage of the concentrated gel is 80V, the working voltage of the separation gel is 120V, and the time is 90 min. And (3) transferring the corresponding strip on the gel to the PVDF membrane by a wet transfer method under the constant current of 100V for 90 min. Then putting the PVDF membrane into 50g/mL skimmed milk powder, sealing at room temperature for 2h, then transferring into prepared ACE2 polyclonal antibody diluted by TBST according to a ratio of 1: 3000, incubating overnight at 4 ℃, washing the membrane for 5 times by TBST, then transferring into goat anti-rabbit IgG labeled by horseradish enzyme diluted by TBST solution according to a ratio of 1: 5000, incubating at room temperature for 2h, and washing by TBST. And observing and taking a picture in a full-automatic chemiluminescence image analysis system after color development.
The results are shown in fig. 10, ACE2 is expressed in heart, liver, kidney, jejunum, ileum, duodenum, cecum, and rectum, and the expression level in heart, ileum, rectum, and jejunum is higher.
(2) Western Blot identification of antibody specificity of different tissues of chicken, sheep, pig and rat
Western Blot analysis and identification are carried out by taking the prepared ACE2 polyclonal antibody as a primary antibody and goat anti-mouse IgG-HRP as a secondary antibody and taking chicken ileum, chicken duodenum, sheep ileum, sheep duodenum, rat ileum, rat duodenum, pig ileum and pig duodenum proteins as secondary antibodies, and the results are shown in figure 11, specific bands appear in the chicken ileum and the duodenum at 100kDa, and no specific band appears in the sheep ileum, the sheep duodenum, the rat ileum, the rat duodenum, the pig ileum and the pig duodenum. The results show that the polyclonal antibody prepared by the invention can specifically recognize ACE2 protein in chicken tissues, but cannot well recognize ACE2 protein in tissues of other species, namely the prepared chicken anti-mouse ACE2 polyclonal antibody has better specificity.
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 such modifications and substitutions do not depart from the spirit and scope of the present invention as defined by the appended claims.
Claims (10)
1. An ACE2 protein of chicken, which is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
2. A gene encoding ACE2 protein of chicken as described above, wherein the gene encodes the amino acid sequence of the protein of claim 1, or the nucleotide sequence of the gene is shown in SEQ ID No. 2.
3. A connection vector or an expression vector of chicken ACE2, which is characterized in that the connection vector or the expression vector contains a nucleotide sequence shown as SEQ ID NO. 2.
4. A preparation method of chicken ACE2 cloning vector or expression vector is characterized by comprising 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. Genomic RNA was extracted and the RNA was reverse transcribed into cDNA using random primers. Designing an upstream primer and a downstream primer of ACE2 for ACE2 full-length gene amplification, respectively adding BamHI and HindIII enzyme cutting sites on two sides of an ACE2 gene fragment, and taking cDNA as a template to obtain the ACE2 gene full length by amplification.
S2, cutting glue to purify the target fragment, and then connecting the target fragment to a pMD19T vector by using a TA cloning method to obtain a plasmid pMD19T-ACE 2.
5. The method of claim 4, further comprising the steps of:
s3, digesting the pET32a vector and the pMD19T-ACE2 plasmid respectively by BamHI and HindIII, cutting gel to recover a target fragment, and connecting the target fragment to the vector by a T4 connection method to obtain the plasmid pET32a-ACE 2.
6. The method of claim 4, wherein in step S1, the ACE2 primer has the sequence shown in SEQ ID No.3 and SEQ ID No.3, and the amplification conditions are as follows: 5min at 95 ℃; 20s at 95 ℃, 20s at 59 ℃, 1min at 72 ℃ for 15s, and 40 cycles; 10min at 72 ℃.
7. The method according to claim 4, wherein in step S2, the specific linkage system: 0.5uL of pMD19-T vector, 4.5uL of purified product and 5uLSolution I are put in a 200uLEP tube, mixed evenly and run on a PCR instrument for 16 ℃ for 14h to be connected.
8. The method according to claim 5, wherein in step S3, the specific steps are: connecting the recovered product of the pET32a vector with the recovered product of the pMD-19T-ACE2 recombinant plasmid by using T4 ligase, and connecting the obtained products to a system (10 uL); 1uL pET32a vector, 7uL target sequence, 1uLT4 ligase, 1uL10 xbuffer in 200uLEP tube, mixing, in PCR instrument at 16 degrees C, 14h, connecting, get plasmid pET32a-ACE 2.
9. A method for preparing chicken ACE2 protein comprises transferring chicken ACE2 prokaryotic expression vector prepared in claim 5 into DH5 alpha and BL21 competent cells, performing IPTG induced expression, and performing KCL gel cutting purification to obtain pET32a-ACE2 recombinant protein.
10. The preparation of polyclonal antibody against ACE2 protein includes the following steps: the chicken pET32a-ACE2 recombinant protein obtained in the claim 9 is used for immunizing Wistar rats, the inoculation mode is subcutaneous multipoint injection at the back of the neck, a Freund complete adjuvant is used for a first immunity, a Freund incomplete adjuvant is used for a first immunity, a second immunity, a third immunity and a fourth immunity respectively, and rat serum is taken 7 days after the boosting immunity.
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Non-Patent Citations (4)
| Title |
|---|
| JI,X.X.等: ""Gallus gallus ACE2 (ACE2) mRNA, complete cds"， GenBank: MK560199.1" * |
| 徐春生;姚一琳;杨平;JAMEEL AHMED GANDAHI;包慧君;卞勋光;邬丽;陈秋生;: "中华鳖肾脏ACE2基因克隆及其遗传进化分析" * |
| 杨维维: "山羊血管紧张素转换酶2(ACE2)的克隆表达及抗体制备" * |
| 肖航;王凯;王换换;王鲲;张源淑;: "真核表达质粒pcDNA.3.1(+)-ACE2的构建及其在CHO细胞中的表达" * |
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