CN111876438A - Salmonella typhi recombinant fusion protein HlyE-CdtB, coding gene thereof, expression and application thereof - Google Patents
Salmonella typhi recombinant fusion protein HlyE-CdtB, coding gene thereof, expression and application thereof Download PDFInfo
- Publication number
- CN111876438A CN111876438A CN202010646028.8A CN202010646028A CN111876438A CN 111876438 A CN111876438 A CN 111876438A CN 202010646028 A CN202010646028 A CN 202010646028A CN 111876438 A CN111876438 A CN 111876438A
- Authority
- CN
- China
- Prior art keywords
- hlye
- cdtb
- fusion protein
- salmonella typhi
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/255—Salmonella (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/255—Salmonella (G)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention relates to the field of genetic engineering, in particular to a salmonella typhi recombinant fusion protein HlyE-CdtB, a coding gene thereof, and expression and application thereof. The salmonella typhi recombinant fusion protein HlyE-CdtB is recombinantly expressed in an escherichia coli expression system, and the salmonella typhi recombinant fusion protein HlyE-CdtB has the advantages of short production period, high yield, low cost and good specificity; the His label on the recombinant protein is beneficial to one-step purification to achieve higher purity, and can show better activity without enzyme digestion; the recombinant antigen can be used as a part of a canine parvovirus colloidal gold detection reagent strip, has high sensitivity, good specificity, good stability and simple and convenient operation, and is suitable for being used as a conventional detection means.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a salmonella typhi recombinant fusion protein HlyE-CdtB, a coding gene thereof, and expression and application thereof.
Background
Salmonella is a common gram-negative bacterium causing zoonosis, belonging to the family enterobacteriaceae. The name was isolated from hog cholera as early as 1885 by TheobaldSmith and DanielElmerSalmon. Salmonella antigens are complex, and serotypes based on O antigens (parietal antigens) and H antigens (flagellar antigens) are typed over 3000. Most of these serotypes are capable of cross-infecting between humans and animals and can cause a range of diseases of varying severity, from mild diarrhea to severe sepsis. The genus Salmonella includes two major species, Salmonella enterica and Salmonella indigenous. Can be divided into 7 subtypes: i, II, IIIa, IIIb, IV, VI and V, the former type 6 belongs to s.enterica and s.bongori belongs to subtype V. They are 95% to 99% DNA sequence similar. The host of subtype I (Salmonella enterica) is mainly mammals and birds, which contain important medical pathogens. According to the clinical syndrome of human caused by salmonella, the salmonella can be simply defined as two types: salmonella typhi and non-salmonella typhi.
The pathogenicity of salmonella is strongly related to its virulence factors. Many pathogenicity-related genes have been identified by whole genome sequencing, which involves primarily numerous virulence factors, including adhesion factors, siderophores, capsules, endotoxins (LPS), exotoxins, invasins, and type iii and type iv secretion systems. The pathogenicity of salmonella is primarily controlled by these virulence genes. Most of these virulence factors that can elicit an immune inflammatory response in a host are identified and tested in animal models. These virulence factors that cause infection fall into two major categories, those associated with pathogens that stimulate the innate immune system and those associated with virulence that use the host inflammatory process to cause pathological changes. Another class of virulence factors are toxin genes, such as the cdt gene, which encode a cell-swelling toxin that causes slow swelling death in eukaryotic cells. cdtB, among salmonella, was first found in salmonella typhi and salmonella paratyphi, acting together with pltA, pltB, causing apoptosis and necrosis of cells, which may be associated with infection with salmonella typhi.
After people and livestock are infected with salmonella, symptoms can be aggravated or the death rate can be increased, the animal fertility can be reduced, great economic loss is caused, the health of people and the health development of animal husbandry are seriously threatened, and the prevention and the treatment of the salmonella and the animal fertility are an important subject of public health. The method can quickly, accurately and simply detect the salmonella, and has important significance in the aspects of medical health, food health, animal epidemic disease monitoring and the like. The salmonella detection is mainly performed by the traditional detection method, and the conventional methods such as non-selective and selective enrichment, suspicious bacteria separation and the like are classic and reliable, but have complex and very complicated procedures, time and labor waste, poor sensitivity and specificity and high omission ratio. There are also some methods such as immunofluorescence, enzyme-linked immunosorbent assay (ELISA), Polymerase Chain Reaction (PCR) techniques, such as "an ELISA kit for detecting salmonella antibodies" published under CN106290918A, including: solid phase carrier coated with recombinant protein PagC, enzyme-labeled antibody, salmonella negative serum and positive serum. The ELISA kit and the method for detecting the salmonella antibody are widely applicable to salmonella, have wide application range, have higher specificity and repeatability, but need to use specified instruments and equipment, have corresponding test conditions and skills, are difficult to popularize in a basic layer, have long detection time, can detect results in hours, have poor labeling stability, and are not suitable for conventional detection means.
Disclosure of Invention
The invention aims to overcome the defects of high requirement on equipment, long detection time and unsuitability for conventional detection of the existing detection method, and provides the salmonella typhi recombinant fusion protein HlyE-CdtB, the coding gene thereof, the expression and the application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a salmonella typhi recombinant fusion protein HlyE-CdtB gene has a sequence shown in SEQ ID NO. 2.
The recombinant fusion protein HlyE-CdtB of the salmonella typhi coded by the gene is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.
The recombinant fusion protein HlyE-CdtB Gene of the salmonella typhi is designed according to the protein sequence of NCBI Gene bank NC-003277.2, and the most dominant epitope in the recombinant fusion protein is selected for fusion by analyzing the epitope on the recombinant fusion protein.
A recombinant vector comprising the recombinant antigen gene.
Preferably, the vector is an escherichia coli BL21 expression vector. The recombinant protein gene is artificially synthesized, the used vector is pET30a which is kanamycin resistance, a histidine tag is expressed in a fusion mode, and the protein is positioned in the periplasm of cells.
A recombinant strain comprising the above antigen gene.
A method for expressing a salmonella typhi recombinant fusion protein HlyE-CdtB comprises the following steps
(1) Transforming the recombinant vector into escherichia coli BL21 cells to obtain a recombinant strain;
(2) the recombinant strain is cultured in an LB culture medium at the temperature of 35-37 ℃ in a shaking way until OD =0.5-0.7, 0.8-1.2mM IPTG is added, and the induction expression is carried out for 3-5 hours at the temperature of 35-37 ℃ and at the speed of 200-250 rpm;
(3) and after induction, centrifugally recovering, breaking bacteria, taking supernatant and purifying the expressed salmonella typhi recombinant fusion protein HlyE-CdtB.
The expression system of the invention is an escherichia coli BL21 expression system, and has the characteristics of short period, low cost, large expression quantity and the like; in order to better maintain the active site of the recombinant protein, the invention selects a relatively mild culture and induction condition, the induction temperature is 35-37 ℃ during expression, the induction speed is 200-250rpm, and the concentration of induced IPTG is 0.8-1.2mM, so that the recombinant protein has slower expression and sufficient time for spatial conformation formation.
Preferably, the bacteria breaking is carried out by using an ultrasonic breaking mode, wherein the conditions are 350-450w, 2-4s of ultrasonic treatment and 4-8s of interval, and the total time is 150-200 times; the purification method is affinity chromatography, the buffer system is 40-60mM Tris with pH =7.8-8.2, 0.15-0.25M NaCl, and 0.0.4-0.6M Imidazole is added into the eluent; after the purification, the mixture was dialyzed 3 to 5 times in the same buffer system as that used for the purification and sterile-filtered, and the concentration was measured by BCA method. The His tag is added into the recombinant protein, the high purity can be achieved through one-step purification, the high activity can be shown without enzyme digestion after the high-purity recombinant protein is obtained, and compared with the recombinant protein which can only generate the activity through enzyme digestion, the method has the advantages that the steps are reduced, and the cost is reduced.
The protein recombinant fusion protein HlyE-CdtB is applied to preparation of a colloidal gold detection reagent strip.
Preferably, the preparation steps of the colloidal gold test reagent strip are as follows:
(1) adding chloroauric acid and trisodium citrate into boiling pure water, continuously heating and boiling for 10-20min, and cooling to room temperature to obtain colloidal gold solution;
(2) adding K into the cooled colloidal gold solution2CO3Adjusting the pH to be 6.8-7.2, adding the salmonella typhi recombinant fusion protein HlyE-CdtB, stirring for 20-40min, adding a BSA solution, stirring for 20-40min, centrifuging to obtain a precipitate, diluting the precipitate with a gold-labeled diluent, soaking glass fibers, and freeze-drying to obtain a gold-labeled pad;
(3) diluting proteinA to 0.8-1.2mg/mL with a dot-film diluent, diluting goat anti-OspC-VlsE polyclonal antibody to 0.4-0.6mg/mL with the same diluent, streaking the two diluted solutions onto a nitrocellulose membrane, and drying at 35-37 ℃ overnight, wherein the dot-film diluent is 10-20mM PBS (pH 7.2-7.5) and 0.8-1.2wt% sucrose solution;
(4) and assembling the gold label pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the colloidal gold detection reagent strip.
The colloidal gold detection reagent strip prepared by the invention is an indirect colloidal gold detection reagent strip, and compared with other detection modes, the indirect colloidal gold detection reagent strip has the advantages of convenience, rapidness, specificity, sensitivity, strong stability, no need of special equipment and reagents, intuitive result judgment and the like, and is suitable for being used as a conventional detection means.
Preferably, in the preparation method: the gold-labeled diluent is 10-20mM Tris with pH =7.8-8.2, 0.15-0.25M NaCl, 0.5-1.5wt% BSA, 0.1-0.2wt% TritonX-100 and 0.01-0.02wt% Proclin300 solution; the addition amount of the chloroauric acid is 0.01-0.02wt%, and the addition amount of the trisodium citrate is 0.01-0.02 wt%.
In conclusion, the invention has the following beneficial effects: (1) the salmonella typhi recombinant fusion protein HlyE-CdtB is recombinantly expressed in an escherichia coli expression system, and the salmonella typhi recombinant fusion protein has the advantages of short production period, high yield, low cost and good specificity; (2) the His label on the recombinant protein is beneficial to one-step purification to achieve higher purity, and can show better activity without enzyme digestion; (3) the recombinant fusion protein HlyE-CdtB can be used as a part of a salmonella typhi colloidal gold detection kit, has high sensitivity, good specificity, good stability and simple and convenient operation, and is suitable for being used as a conventional detection means.
Drawings
FIG. 1 shows the test results of the test strip of the present invention, in which the first two strips from left to right are positive and the last three strips are negative.
Detailed Description
Example 1: construction of recombinant fusion protein HlyE-CdtB gene expression vector containing salmonella typhosa
The recombinant fusion protein HlyE-CdtB Gene of the salmonella typhi is designed according to the protein sequence of NCBI Gene bank NC-003277.2, the epitope on the recombinant fusion protein is analyzed, and the most dominant epitope is selected for fusion.
The nucleotide sequence obtained by reverse translation of the amino acid sequence into a nucleotide sequence using the codon of E.coli was synthesized by the general biological System (Anhui) Ltd as a recombinant gene sequence SEQ ID NO.2, and the vector was pET30 a.
Example 2: expression of recombinant fusion protein HlyE-CdtB containing salmonella typhi
The synthesized recombinant fusion protein HlyE-CdtB plasmid of the salmonella typhi is transformed into escherichia coli BL21, coated on an LB plate containing 50ug/mL kanamycin (Shanghai, product number: K0408), cultured overnight at 37 ℃, a monoclonal colony is picked up, cultured by 300mL LB medium containing the same concentration of kanamycin at 37 ℃ until OD600 reaches about 0.6, and induced and expressed by IPTG (Shanghai, product number: IB0168) with the final concentration of 1mM, and the induction conditions are as follows: at 37 ℃ and at a rotation speed of 200rpm for 4 h. After induction, the culture was centrifuged at 4 ℃ and 7000rpm for 10min to collect the cells.
Example 3: purification and renaturation of recombinant fusion protein HlyE-CdtB containing salmonella typhi
The cells were disrupted with 50mL Binding Buffer consisting of 50mM Tris, 0.2M NaCl, pH =8.0, and then sonicated at 400w for 3s with 6s intervals for 180 times, and finally centrifuged at 12000rpm for 30min at 4 ℃ to collect the supernatant, which was the target protein. Followed by one-step purification on a Ni + column, eluting the protein of interest with an Elution Buffer consisting of 50mM Tris, 0.2M NaCl, 0.5M imidazole, pH = 8.0. The purified recombinant protein was dialyzed against dialysis buffer consisting of 50mM Tris, 0.2M NaCl, pH =8.0, and the dialysate was changed every 12h for 3 times. The dialyzed protein solution was taken out, filtered through a 0.22um filter, measured for concentration by BCA method, and stored at-20 ℃ for further use.
Example 4: indirect gold-labeled method for detecting salmonella typhi antibody
4.1 preparation of Salmonella typhi colloidal gold detection reagent strip
Adding 1000mL of ultrapure water into a triangular flask, heating the ultrapure water on a magnetic heating stirrer until the ultrapure water is boiled, then adding 1mL of 10% chloroauric acid (sigma), then adding 1mL of 10wt% trisodium citrate solution, continuing heating and boiling for 15min, and then cooling to room temperature;
putting 100mL of cooled colloidal gold solution into a beaker, and adding 0.2M K into the beaker with stirring2CO31.5mL of the mixed solution is adjusted to the pH =7.0 of gold water, a certain amount of salmonella typhi antibody recombinant fusion protein HlyE-CdtB is added after stirring, the mixed solution is stirred for 30min at room temperature, 10mL of 10wt% BSA solution is added, centrifugation is carried out for 30min at 12000rpm after stirring for 30min at room temperature, the supernatant is carefully sucked out and discarded, and the precipitate is diluted to 1mL by using a gold-labeled diluent which comprises 20mM Tris, 0.2M NaCl, 1wt% BSA, 0.1wt% TritonX-100, 0.01wt% Proclin300 and pH =8.0, so that the volume is determined to be 1mL, and the mixed solution is a labeled salmonella typhi antigen colloidal gold compound;
diluting the gold-labeled compound by 100 times with a gold-labeled diluent, soaking glass fiber in the diluted gold-labeled compound, and freeze-drying the glass fiber to obtain a gold-labeled pad;
diluting proteinA to 1.0mg/mL with spot membrane diluent of composition 10mM PBS, 1wt% sucrose, pH =7.4, diluting goat anti-OspC-VlsE polyclonal antibody to 0.5mg/mL with the same diluent, streaking the two diluted solutions onto nitrocellulose membrane, oven drying at 37 ℃ overnight;
and assembling the gold-labeled pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the salmonella typhi colloidal gold detection reagent strip.
4.2 detection of Salmonella typhi colloidal gold detection reagent strip
Adding 50ul of samples (serum and plasma) to be detected to a sample pad, standing at room temperature for 10min, and determining the result according to the following result determination standards:
firstly, only one strip appears on the quality control line, no strip appears in the test area, and the test area is negative;
two strips appear, wherein one strip is positioned in the quality control area, and the other strip is positioned in the test area and is positive;
and thirdly, the quality control line has no strip, which indicates that the test strip is damaged, and the test strip should be replaced with a new test strip for retesting.
The detection results are shown in FIG. 1, wherein the first two are positive and the last three are negative from left to right, and it can be clearly seen from the figure that only one band appears on the quality control line of the sample with the negative result, and no band appears in the test zone; while the first band from left to right is lighter, it can be seen that there are two bands of positive results, one in the control zone and the other in the test zone.
4.3 Salmonella typhi colloidal gold detection reagent strip salmonella typhi antibody detection result
The salmonella typhi colloidal gold detection reagent strip is used for detecting 100 parts of positive salmonella typhi patient serum and 400 parts of normal human serum, wherein 98 positive cases are detected in 100 parts of positive serum, 2 negative cases are missed, 5 false positives appear in 400 parts of negative serum, and the sensitivity and specificity are respectively 98% and 98.5%, and the result data can show that the salmonella typhi recombinant fusion protein HlyE-CdtB has very good sensitivity and specificity, is greatly increased compared with the 90-95% sensitivity and specificity of the existing colloidal gold detection reagent strip, and is very suitable for being used as a raw material for manufacturing the salmonella typhi antibody detection test strip; the prepared colloidal gold detection strip has the advantages of high sensitivity, good specificity, short detection time, good stability, simple and convenient operation and the like, can judge a result only by placing the strip at room temperature for 10 minutes, and is very suitable for being used as a conventional detection method.
Example 5: expression of recombinant fusion protein HlyE-CdtB containing salmonella typhi
The synthesized recombinant fusion protein HlyE-CdtB plasmid of the salmonella typhi is transformed into escherichia coli BL21, coated on an LB plate containing 50ug/mL kanamycin (Shanghai, product number: K0408), cultured overnight at 35 ℃, a monoclonal colony is picked up, cultured by 300mL LB medium containing the same concentration of kanamycin at 35 ℃ until OD600 reaches about 0.7, and induced and expressed by IPTG (Shanghai, product number: IB0168) with the final concentration of 1.2mM, and the induction conditions are as follows: at 35 ℃ and at 250rpm for 3 h. After induction, the culture was centrifuged at 4 ℃ and 7000rpm for 10min to collect the cells.
Example 6: purification and renaturation of recombinant fusion protein HlyE-CdtB containing salmonella typhi
The cells were disrupted with 50mL Binding Buffer consisting of 40mM Tris, 0.15M NaCl, pH =8.2, and then sonicated under conditions of 450w, sonication for 2s, 4s intervals, 200 times total, and finally, at 12000rpm, 30min, the supernatant was collected by centrifugation at 4 ℃ and the target protein was in the supernatant. Followed by one-step purification on a Ni + column, eluting the protein of interest with an Elution Buffer consisting of 40mM Tris, 0.15M NaCl, 0.4M imidazole, pH = 8.2. The purified recombinant protein was dialyzed against dialysis buffer consisting of 40mM Tris, 0.15M NaCl, pH =8.2, and the dialysate was changed every 12h for 4 times. The dialyzed protein solution was taken out, filtered through a 0.22um filter, measured for concentration by BCA method, and stored at-20 ℃ for further use.
Example 7: preparation of salmonella typhi colloidal gold detection reagent strip
Adding 1000mL of ultrapure water into a triangular flask, heating the ultrapure water on a magnetic heating stirrer until the ultrapure water is boiled, then adding 1mL of 10% chloroauric acid (sigma), then adding 1mL of 10wt% trisodium citrate solution, continuing heating and boiling for 10min, and then cooling to room temperature;
putting 100mL of cooled colloidal gold solution into a beaker, and adding 0.2M K into the beaker with stirring2CO31.5mL of the mixed solution is adjusted to the pH =7.2 of the gold water, a certain amount of the salmonella typhi recombinant fusion protein HlyE-CdtB is added after stirring, the mixed solution is stirred for 20min at room temperature, 10mL of 10wt% BSA solution is added, centrifugation is carried out for 30min at 12000rpm after stirring for 20min at room temperature, the supernatant is carefully sucked out and discarded, and the precipitate is diluted to 1mL by using a gold-labeled diluent consisting of 10mM Tris, 0.15M NaCl, 1.5wt% BSA, 0.15wt% TritonX-100, 0.015wt% Proclin300 and pH =8.2, so that the volume is determined to be 1mL, and the labeled salmonella typhi colloidal gold compound is obtained;
diluting the gold-labeled compound by 100 times with a gold-labeled diluent, soaking glass fiber in the diluted gold-labeled compound, and freeze-drying the glass fiber to obtain a gold-labeled pad;
diluting proteinA a to 0.8mg/mL with spot membrane diluent consisting of 10mM PBS, 0.8wt% sucrose, pH =7.2, diluting goat anti-OspC-VlsE polyclonal antibody to 0.4mg/mL with the same diluent, streaking the two diluted solutions onto nitrocellulose membrane, oven drying overnight at 35 ℃;
and assembling the gold-labeled pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the salmonella typhi colloidal gold detection reagent strip.
Example 8: expression of recombinant fusion protein HlyE-CdtB containing salmonella typhi
The synthesized recombinant fusion protein HlyE-CdtB plasmid of the salmonella typhi is transformed into escherichia coli BL21, coated on an LB plate containing 50ug/mL kanamycin (Shanghai, product number: K0408), cultured overnight at 36 ℃, a monoclonal colony is picked up, cultured by 300mL LB medium containing the same concentration of kanamycin at 36 ℃ until OD600 reaches about 0.5, and induced and expressed by IPTG (Shanghai, product number: IB0168) with the final concentration of 0.8mM, and the induction conditions are as follows: at 36 deg.C, 230rpm, 5 h. After induction, the culture was centrifuged at 4 ℃ and 7000rpm for 10min to collect the cells.
Example 9: purification and renaturation of recombinant fusion protein HlyE-CdtB containing salmonella typhi
The cells were disrupted with 50mL Binding Buffer consisting of 50mM Tris, 0.2M NaCl, pH =8.0, and then sonicated at 350w for 4s with 8s intervals for 150 times, and finally centrifuged at 12000rpm for 30min at 4 ℃ to collect the supernatant, which was the target protein. Followed by one-step purification on a Ni + column, eluting the protein of interest with an Elution Buffer consisting of 60mM Tris, 0.25M NaCl, 0.5M imidazole, pH = 7.8. The purified recombinant protein was dialyzed against a dialysis buffer consisting of 60mM Tris, 0.25M NaCl, pH =7.8, and the dialysate was changed every 12h for 5 times. The dialyzed protein solution was taken out, filtered through a 0.22um filter, measured for concentration by BCA method, and stored at-20 ℃ for further use.
Example 10: preparation of salmonella typhi colloidal gold detection reagent strip
Adding 1000mL of ultrapure water into a triangular flask, heating the ultrapure water on a magnetic heating stirrer until the ultrapure water is boiled, then adding 1mL of 10% chloroauric acid (sigma), then adding 1mL of 10wt% trisodium citrate solution, continuing heating and boiling for 20min, and then cooling to room temperature;
putting 100mL of cooled colloidal gold solution into a beaker, and adding 0.2M K into the beaker with stirring2CO31.5mL of the mixed solution is adjusted to the pH =6.8 of the gold water, a certain amount of the salmonella typhi recombinant fusion protein HlyE-CdtB is added after stirring, the mixed solution is stirred for 30min at room temperature, 10mL of a 10wt% BSA solution is added, centrifugation is carried out for 30min at 12000rpm after stirring for 30min at room temperature, the supernatant is carefully sucked out and discarded, and the precipitate is diluted to 1mL by using a gold-labeled diluent consisting of 15mM Tris, 0.25M NaCl, 0.5wt% BSA, 0.2wt% TritonX-100, 0.02wt% Proclin300 and pH =7.8, so that the volume is determined to be 1mL, and the labeled salmonella typhi colloidal gold compound is obtained;
diluting the gold-labeled compound by 100 times with a gold-labeled diluent, soaking glass fiber in the diluted gold-labeled compound, and freeze-drying the glass fiber to obtain a gold-labeled pad;
diluting proteinA a to 1.2mg/mL with spot membrane diluent of composition 10mM PBS, 1.2wt% sucrose, pH =7.5, diluting goat anti-OspC-VlsE polyclonal antibody to 0.6mg/mL with the same diluent, streaking the two diluted solutions onto nitrocellulose membrane, oven drying at 36 ℃ overnight;
and assembling the gold-labeled pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the salmonella typhi colloidal gold detection reagent strip.
Claims (10)
1. A salmonella typhi recombinant fusion protein HlyE-CdtB gene is characterized in that the sequence of the recombinant antigen gene is shown in SEQ ID NO. 2.
2. The recombinant fusion protein HlyE-CdtB of Salmonella typhi encoded by the gene of claim 1, wherein the amino acid sequence is represented by SEQ ID No. 1.
3. A recombinant vector comprising the recombinant fusion protein HlyE-CdtB gene of claim 1.
4. The recombinant vector according to claim 3, wherein the vector is an E.coli BL21 expression vector.
5. A recombinant strain comprising the antigenic gene of claim 1.
6. A method for expressing a salmonella typhi recombinant fusion protein HlyE-CdtB is characterized by comprising the following steps:
(1) transforming the recombinant vector of claim 4 into Escherichia coli BL21 cells to obtain a recombinant strain;
(2) the recombinant strain is cultured in an LB culture medium at the temperature of 35-37 ℃ in a shaking way until OD =0.5-0.7, 0.8-1.2mM IPTG is added, and the induction expression is carried out for 3-5 hours at the temperature of 35-37 ℃ and at the speed of 200-250 rpm;
(3) and after induction, centrifugally recovering, breaking bacteria, taking supernatant and purifying the expressed salmonella typhi recombinant fusion protein HlyE-CdtB.
7. The method as claimed in claim 6, wherein the disruption is performed by ultrasonic disruption under conditions of 350-450w, 2-4s of ultrasonic treatment, and 4-8s of interval, and for a total of 150-200 times; the purification method is affinity chromatography, the buffer system is 40-60mM Tris with pH =7.8-8.2, 0.15-0.25M NaCl, and 0.4-0.6M Imidazole is added into the eluent; after the purification, the mixture was dialyzed 3 to 5 times in the same buffer system as that used for the purification and sterile-filtered, and the concentration was measured by BCA method.
8. The recombinant fusion protein HlyE-CdtB of claim 2, which is used for preparing a colloidal gold detection reagent strip.
9. The use of claim 8, wherein the colloidal gold test strip is prepared by the following method:
(1) adding chloroauric acid and trisodium citrate into boiling pure water, continuously heating and boiling for 10-20min, and cooling to room temperature to obtain colloidal gold solution;
(2) adding K into the cooled colloidal gold solution2CO3Adjusting the pH to be 6.8-7.2, adding the salmonella typhi recombinant fusion protein HlyE-CdtB, stirring for 20-40min, adding a BSA solution, stirring for 20-40min, centrifuging to obtain a precipitate, diluting the precipitate with a gold-labeled diluent, soaking glass fibers, and freeze-drying to obtain a gold-labeled pad;
(3) diluting proteinA to 0.8-1.2mg/mL with a dot-film diluent, diluting goat anti-OspC-VlsE polyclonal antibody to 0.4-0.6mg/mL with the same diluent, streaking the two diluted solutions onto a nitrocellulose membrane, and drying at 35-37 ℃ overnight, wherein the dot-film diluent is 10-20mM PBS (pH 7.2-7.5) and 0.8-1.2wt% sucrose solution;
(4) and assembling the gold label pad, the coated nitrocellulose membrane, filter paper, a polyester plate, a sample pad and the like into the colloidal gold detection reagent strip.
10. Use according to claim 9, characterized in that in the preparation method: the gold-labeled diluent is 10-20mM Tris with pH =7.8-8.2, 0.15-0.25M NaCl, 0.5-1.5wt% BSA, 0.1-0.2wt% TritonX-100 and 0.01-0.02wt% Proclin300 solution; the addition amount of the chloroauric acid is 0.01-0.02wt%, and the addition amount of the trisodium citrate is 0.01-0.02 wt%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010646028.8A CN111876438A (en) | 2020-07-07 | 2020-07-07 | Salmonella typhi recombinant fusion protein HlyE-CdtB, coding gene thereof, expression and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010646028.8A CN111876438A (en) | 2020-07-07 | 2020-07-07 | Salmonella typhi recombinant fusion protein HlyE-CdtB, coding gene thereof, expression and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111876438A true CN111876438A (en) | 2020-11-03 |
Family
ID=73150314
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010646028.8A Pending CN111876438A (en) | 2020-07-07 | 2020-07-07 | Salmonella typhi recombinant fusion protein HlyE-CdtB, coding gene thereof, expression and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111876438A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112630421A (en) * | 2021-03-05 | 2021-04-09 | 山东康华生物医疗科技股份有限公司 | Method, reagent strip and kit for labeling salmonella typhi recombinant antigen with colloidal gold |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106093409A (en) * | 2016-06-07 | 2016-11-09 | 江南大学 | The preparation method of the colloidal gold strip that detection Salmonella in Food based on Salmonella core polysaccharide monoclonal antibody belongs to |
| CN106290918A (en) * | 2016-08-24 | 2017-01-04 | 南京农业大学 | A kind of ELISA kit detecting antibodies toward salmonella |
-
2020
- 2020-07-07 CN CN202010646028.8A patent/CN111876438A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106093409A (en) * | 2016-06-07 | 2016-11-09 | 江南大学 | The preparation method of the colloidal gold strip that detection Salmonella in Food based on Salmonella core polysaccharide monoclonal antibody belongs to |
| CN106290918A (en) * | 2016-08-24 | 2017-01-04 | 南京农业大学 | A kind of ELISA kit detecting antibodies toward salmonella |
Non-Patent Citations (1)
| Title |
|---|
| 王小龙等: "沙门氏菌invH基因的重组表达与免疫保护效果研究", 《中国兽医科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112630421A (en) * | 2021-03-05 | 2021-04-09 | 山东康华生物医疗科技股份有限公司 | Method, reagent strip and kit for labeling salmonella typhi recombinant antigen with colloidal gold |
| CN112630421B (en) * | 2021-03-05 | 2021-06-01 | 山东康华生物医疗科技股份有限公司 | Method, reagent strip and kit for labeling salmonella typhi recombinant antigen with colloidal gold |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115043948B (en) | Monkey pox virus specific fusion protein antigen and preparation method and application thereof | |
| CN107176977B (en) | Application of mycoplasma bovis Mbovp730 protein in natural infection and vaccine immune identification | |
| Tang et al. | Diversity of Helicobacter pylori isolates in expression of antigens and induction of antibodies | |
| CN107304231B (en) | Mycobacterium tuberculosis fusion protein and application thereof | |
| CN110642927A (en) | Application of protein in preparation of medicine for preventing cryptococcus pyogenes infection | |
| CN111621506B (en) | Mycoplasma bovis secretory protein Mbovp0145 and application thereof | |
| CN111337668A (en) | Novel coronavirus antigen colloidal gold rapid diagnosis kit and preparation method thereof | |
| CN111876438A (en) | Salmonella typhi recombinant fusion protein HlyE-CdtB, coding gene thereof, expression and application thereof | |
| RU2260047C2 (en) | Recombinant plasmid dna providing synthesis of borrelia garinii immunodominant protein for lyme-borreliosis diagnosis | |
| CN108956985B (en) | Indirect ELISA (enzyme-linked immunosorbent assay) detection kit for detecting novel goose astrovirus antibody and application | |
| CN111548423B (en) | Mycoplasma pneumoniae fusion antigen and preparation method and application thereof | |
| CN111875677A (en) | Canine parvovirus VP2 protein recombinant antigen, encoding gene thereof, expression and application thereof | |
| CN116445448B (en) | Acinetobacter baumannii PLPFP recombinant protein, preparation method and application | |
| JP5183635B2 (en) | In vitro diagnostic method for PVL-producing Staphylococcus aureus | |
| CN116478953B (en) | Acinetobacter baumannii DlaT recombinant protein, preparation method and application | |
| CN108101974B (en) | Fasciola hepatica multi-epitope fusion diagnostic antigen and application and preparation method thereof | |
| CN111978410A (en) | Fusion protein of brucella outer membrane protein OMP25 and periplasmic protein BP26 as well as expression and application thereof | |
| CN111018998A (en) | Lyme recombinant fusion protein OspC-VlsE and application thereof | |
| CN114288402B (en) | Preparation method and application of mycoplasma hyopneumoniae multi-epitope genetic engineering subunit vaccine based on reverse vaccinology technology | |
| CN110382519A (en) | The completely new recombinant protein antigen of Orientia Tsutsugamushi and the vaccine composition for using above-mentioned recombinant protein antigen | |
| CN112501096A (en) | Construction and application of genetically engineered escherichia coli of a group of parenteral pathogenic escherichia coli glycoprotein conjugate vaccines | |
| CN111398604A (en) | Serological detection method of Buffalo A19-delta VirB12 vaccine | |
| CN113698474A (en) | African swine fever polyclonal antibody and African swine fever antigen detection test strip | |
| CN113444157B (en) | Application of mycoplasma hyorhinis outer membrane protein Mhr _0461 in preparation of mycoplasma hyorhinis or antibody detection kit thereof | |
| CN116462743B (en) | Acinetobacter baumannii translation elongation factor recombinant protein, preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20201103 |