CN117603321A - Recombinant typhoid bacillus flagella antigen and preparation method and application thereof - Google Patents
Recombinant typhoid bacillus flagella antigen and preparation method and application thereof Download PDFInfo
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- 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
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- G01N33/56911—Bacteria
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Abstract
The invention discloses a recombinant typhoid bacillus flagella antigen, a preparation method and application thereof, wherein the amino acid sequence of the recombinant typhoid bacillus flagella antigen is shown as SEQ ID NO.4, and the corresponding coding nucleotide sequence is shown as SEQ ID NO. 3. The recombinant typhoid bacillus flagellin antigen comprises a typhoid bacillus flagellin (H) 145-418aa sequence fragment, and the amino acid sequence of the recombinant typhoid bacillus flagellin antigen is shown as SEQ ID NO. 1. The invention obtains the recombinant expression of the dominant epitope of the salmonella typhi flagella antigen, enhances the positive detection rate of the target antigen in immunodiagnosis application, and eliminates the interference caused by homologous sequences of salmonella; the expression level of the recombinant protein in the escherichia coli is improved through codon optimization; the target gene is inserted into the BamHI/EcoRI sites of the vector, so that the N end and the C end of the expressed protein still contain part of amino acid sequences in the vector, and the N end and the C end are provided with 6 xHis labels, thereby facilitating affinity purification, improving the protein purity, improving the labeling efficiency and enhancing the detection sensitivity.
Description
Technical Field
The invention relates to the technical field of genetic engineering and immunodiagnosis, in particular to a recombinant typhoid bacillus flagella antigen and a preparation method and application thereof.
Background
Typhoid bacillus (salmonella typhi, s.typhi): salmonella typhoid bacteria of enterobacteriaceae; size (0.6-1.0 μm) x (2-3 μm), no spores, generally flagellum, no capsule, most pili, gram-negative bacilli. Typhoid bacillus can cause acute digestive tract infectious diseases, commonly known as typhoid fever. The main symptoms are high fever, abdominal pain, severe diarrhea, headache, rose spots on the body, etc. Intestinal bleeding or perforation is its most serious complication. Typical typhoid disease has a long course of disease, about 3-4 weeks. After some patients recover from the disease, the gallbladder still carries bacteria and is continuously excreted along with the feces, which becomes an important infectious source. The bacteria in the recovery period are generally discharged for 3 weeks to 3 months, and a few people can continuously discharge bacteria for more than 1 year.
The diagnosis of typhoid bacillus is based on clinical symptoms and laboratory diagnosis, and clinical symptoms are often atypical due to the increase of light typhoid and the wide use of antibiotics in recent years. Therefore, early rapid diagnosis is a key and important point in controlling the disease. The current common typhoid diagnosis method comprises the following steps: bacterial culture, hypertrophy assay, polymerase chain reaction and immunological detection methods. The first three methods have the defects of long detection time, low detection rate, high false positive rate, requirement for specific equipment, high cost and the like. The immunological detection method is very widely applied due to the characteristics of simple operation, easy judgment of results, suitability for rapid screening of a large number of samples on site and the like. The method mainly realizes the specific detection of the typhoid bacillus by preparing typhoid bacillus antigens or antibodies.
The typhoid bacillus antigen mainly comprises O antigen (thallus antigen), H antigen (flagella antigen) and Vi antigen (virulence antigen). The O antigen is lipopolysaccharide present on the surface of the cell, and is heat-resistant. The H antigen is a bacterial flagellin. The Vi antigen is a thermolabile acidic polysaccharide polymer which exists on the surface of a small number of bacteria, has weak antigenicity and can prevent the agglutination reaction of the O antigen and the corresponding antibody. The thallus (O) antigen, the flagella (H) antigen and the surface (Vi) antigen of the typhoid bacillus can enable the human body to generate corresponding antibodies. However, the O antigen is mostly extracted by natural culture, and high-purity products are difficult to obtain, and false positive, crossover and the like are easy to generate. Vi is poorly antigenic and unsuitable for use; while typhoid bacillus has whole flagellum, and can stimulate the organism to produce early and high titer antibody. Therefore, the H antigen is more advantageous for use as a raw material for immunodiagnosis. However, salmonella flagellin has high homology, and cross false positive is easy to generate in natural extraction.
Therefore, the raw materials with high specificity and high sensitivity are urgently needed in the market, and further research and development are needed.
Disclosure of Invention
Aiming at the problems, the invention provides a recombinant typhoid bacillus flagella antigen and a preparation method and application thereof. The salmonella flagellin has high homology, and cross false positive is easy to generate by natural extraction, so the invention selects a typhoid bacillus species specific region and an epitope rich region to prepare high-specificity and high-sensitivity recombinant H antigen by avoiding a homologous region, can enhance the positive detection rate of a target antigen in immunodiagnosis application, and eliminates interference caused by homologous sequences of salmonella.
Specifically, the technical scheme provided by the application is as follows:
in a first aspect, the present application provides a recombinant typhoid bacillus flagella antigen, the amino acid sequence of which is shown as SEQ ID NO. 4.
In a second aspect, the present application further provides a recombinant typhoid bacillus flagellin, wherein the corresponding coding nucleotide sequence of the recombinant typhoid bacillus flagellin is shown in SEQ ID No. 3.
Further, the amino acid sequence SEQ ID NO.4 comprises an expression vector and a fragment of the sequence of the Salmonella typhi flagellin (H) 145-418 aa.
Furthermore, the sequence fragment of the typhoid bacillus flagellin (H) 145-418aa is shown as SEQ ID NO. 1.
In a third aspect, the present application further provides a recombinant expression vector, which is formed by recombining the expression vector and the codon-optimized nucleotide sequence of the recombinant typhoid bacillus flagella antigen, wherein the codon-optimized nucleotide sequence is shown as SEQ ID No. 2.
The sequence SEQ ID NO.2 is a sequence based on an escherichia coli expression system after codon optimization, does not contain rare codons of escherichia coli, and is suitable for expression in the escherichia coli expression system.
In other cases, in order to express the typhoid bacillus flagellin (H) 145-418aa with the amino acid sequence shown in SEQ ID NO.1 in other prokaryotic or eukaryotic systems, the gene sequence of the typhoid bacillus flagellin can be correspondingly optimized based on the conventional technical means in the field, so that the corresponding coding sequence is obtained.
Therefore, the recombinant typhoid bacillus flagella antigen protected by the application is not limited to a sequence with a coding gene sequence shown as SEQ ID NO. 2.
Furthermore, the recombinant expression vector is obtained by introducing BamH I and EcoR I restriction sites into the 5 'end and 3' end of the coding gene nucleotide sequence SEQ ID NO.2 obtained by optimizing the codon of the second aspect, and then connecting a target gene shown in SEQ ID NO.2 into a prokaryotic expression vector pET28a through gene synthesis.
Preferably, the expression vector is a pET series expression vector, such as pET-32a plasmid, pET-28a or pET-41a, etc.
Under other conditions, the gene sequence is correspondingly optimized based on the conventional technical means in the field, so that the corresponding coding sequence is obtained, and the expression vector can adopt an expression vector suitable for transforming other prokaryotic or eukaryotic expression systems.
In a fourth aspect, the present application further provides a recombinant engineering bacterium prepared by transforming the recombinant expression vector into a host strain.
Preferably, the host bacterium is E.coli, such as E.coli BL21 (DE 3), E.coli Rosseta or other E.coli mutant expression strains, and the like.
Under other conditions, the gene sequence is correspondingly optimized based on the conventional technical means in the field, so that a corresponding coding sequence is obtained, an expression vector suitable for transforming other prokaryotic or eukaryotic expression systems is adopted as an expression vector, and escherichia coli Rosseta or other escherichia coli mutant expression strains, pichia pastoris and the like matched with the expression vector can be adopted as host bacteria.
In a fifth aspect, the present application further provides a method for preparing the recombinant typhoid bacillus flagella antigen, comprising the following steps:
s1, selecting a typhoid bacillus flagellin 145-418aa fragment through sequence homology and antigenicity analysis, obtaining a nucleotide sequence for translating the amino acid through codon optimization, introducing BamHI and EcoRI enzyme cutting sites into the 5 'end and the 3' end, and connecting a target gene shown in SEQ ID NO.2 into a prokaryotic expression vector pET28a through gene synthesis to successfully obtain a recombinant vector pET28a-rH;
s2, transforming the recombinant expression vector into host bacteria, and screening to obtain recombinant engineering bacteria;
s3, culturing and inducing the recombinant engineering bacteria to express, and separating and purifying the product to obtain the recombinant typhoid bacillus flagella antigen.
In a sixth aspect, the present application also provides the use of the recombinant typhoid bacillus flagella antigen in an immunodiagnostic kit.
A typhoid bacillus antibody detection kit, wherein the diagnosis antigen adopts the recombinant typhoid bacillus flagella antigen according to the first aspect.
Preferably, the detection kit is a colloidal gold detection kit.
The invention has the following advantages:
1. the recombinant expression of the dominant epitope of the typhoid bacillus flagella antigen is obtained, the positive detection rate of the target antigen in immunodiagnosis application can be enhanced, and the interference caused by homologous sequences of salmonella is eliminated;
2. according to the invention, the nucleotide sequence of the H antigen fragment is obtained through codon optimization, so that the expression level of the recombinant protein in escherichia coli is improved;
3. according to the invention, the target gene is inserted into BamHI/EcoRI sites of a polyclonal site of the pET28a vector, so that the N end and the C end of the expressed protein still contain part of amino acid sequences in the pET28a vector, the N and C ends have 6-xHis labels, the affinity purification is easy, the protein purity is improved, and meanwhile, the expression of the additional amino acids at the two ends enables the antigen to be used as a marking raw material, the marking efficiency is improved, and the detection sensitivity is enhanced.
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. It is apparent that the drawings in the following description are only one embodiment of the present invention, and that other embodiments of the drawings may be derived from the drawings provided without inventive effort for a person skilled in the art.
Fig. 1: example 1 Anthent assay protein of the invention is hydrophilic, soluble, antigenic, and the like;
wherein 1 and 3 are predicted to be antigenicity, 2 are predicted to be hydrophobicity, 4 are predicted to be hydrophilicity, 5 are predicted to be spiral membrane region, and 6 are predicted to be solubility. The red vertical line indicates the amino acid position, at the current position 146 aa;
fig. 2: the invention of example 1IEDB predicts linear epitope rich regions;
fig. 3: the codon optimization of the embodiment 2 of the invention is related to the codon and the adaptation index;
fig. 4: the embodiment 3 of the invention induces the expression and identifies the glue pattern; wherein 1: a Marker;2: full bacterial map before induction; 3: full bacterial map after induction;
fig. 5: SDS-PAGE and SEC-HPLC results after purification in example 4 of the present invention;
fig. 6: the positive quality control of example 6 of the present invention was tested.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments, and the following embodiments are only for illustrating the present invention and not limiting the scope of the present invention in any way. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention. The experimental methods used in the invention are conventional methods unless otherwise specified. Materials, reagents, and the like used in the present invention are commercially available unless otherwise specified. In addition, other terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.
Example 1: typhoid bacillus flagellin antigen epitope selection
With reference to the amino acid sequence of the flagellin (H) Genbank of Salmonella typhi, AAA27067.1, the protein hydrophilicity, solubility, antigenicity and the like are analyzed by using biological software Anthesprot, and the hydrophilicity, the solubility and the antigenicity are positively related in general cases; the hydrophobicity, the helical membrane region, is inversely related to antigenicity.
As a result of analysis, as shown in FIG. 1, the regions of hydrophilicity, antigenicity and solubility which are mainly 10-21aa, 34-45aa, 59-77aa, 95-129aa, 145-263aa, 284-418aa and 455-469aa; while using the online analysis tool IEDB: bepipred Linear Epitope Prediction 2.0.0 analysis of linear epitope rich regions, results as in fig. 2, analysis of major antigen regions: 19-43aa, 56-66aa, 100-111aa, 117-242aa, 246-421aa, 451-469aa, 496-501aa; meanwhile, by BLAST analysis, the sequence homologous to salmonella of other species has high conservation between the N terminal region and the C terminal region of salmonella; the middle region is a typhoid bacillus specific region; selecting the region which has no homology with other Salmonella and the epitope rich region 145-418aa through the comprehensive analysis; the sequence is shown as SEQ ID NO. 1.
Example 2: sequence codon optimization and recombinant vector construction of typhoid bacillus flagella antigen epitope rich region
In order to increase the expression level of recombinant proteins in E.coli, the E.coli preferred codons were used, genbank: the corresponding nucleotides in AAA27067.1, 145-418aa, were codon optimized and the optimization results are shown in FIG. 3. The nucleotide sequence after optimization is shown as SEQ ID No.2, and the codon adaptation index before optimization CAI=0.81; the codon adaptation index cai=0.96 after optimization. (optimal codon adaptation index is 1); the nucleotide sequence was added upstream and downstream with BamHI and EcoRI cleavage site sequences, respectively, while the 3' end EcoRI was preceded by the complement GGCGGTGGTTCTaa: a segment of linker was translated before supplementing the post C-terminal EcoR I site: GGGS, meanwhile, prevents the dislocation of the C terminal translation, ensures the translation of a 6 XHis tag, is synthesized by the Achilles biotechnology Co., ltd, and the synthesized target gene is cloned in a pET28a vector to construct a recombinant vector pET28a-rH.
The recombinant vector target protein reading frame sequence is shown as SEQ ID NO. 3; the translated protein sequence is recombinant typhoid bacillus flagella antigen (rH), and the amino acid sequence is shown as SEQ ID No. 4; the amino acid sequence SEQ ID No.4 comprises an amino acid sequence translated by partial multiple cloning sites on the N-terminal and C-terminal fusion pET28a vector; wherein the amino acid sequence is: MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGST and GGGSKNSSSVDKLAAALEHHHHHH.
Example 3: recombinant engineering bacteria preparation and expression identification
E.coli BL21 (DE 3) competent cells were transformed with the recombinant vector pET28a-rH constructed in example 2, and the E.coli BL 21-rH was spread on LB plates containing kanavidine resistance (50. Mu.g/mL), cultured upside down overnight at 37℃and the strain was picked up on LB liquid medium containing kanavidine resistance (50. Mu.g/mL), and after shaking culture at 37℃for 5 hours, isopropyl thio-. Beta. -D-galactoside (IPTG) (final concentration of 0.5 mmol/L) was added for induction expression for 4 hours, and bacterial pellet was collected to prepare a protein electrophoresis sample.
SDS-PAGE electrophoresis results show that the recombinant protein is successfully expressed, an induced expression gel diagram is shown in figure 4, and the results show that obvious target band expression exists near 35kDa after induction; the recombinant protein expression strain B/pET28a-rH is successfully obtained.
Example 4: recombinant protein expression purification
4.1 induction of expression:
inoculating 10 μl recombinant plasmid expression strain into 5ml LB culture medium containing 50ug/ml kanamycin (Kan) (purchased from Soy pal), shaking culturing at 37deg.C and 200rpm for overnight (about 16 h), adding 200 μl bacterial liquid into 60ml LB culture medium containing Kan with the same concentration, shaking culturing at 37deg.C and 200rpm for overnight, inoculating all bacterial liquid into 1L LB culture medium containing Kan with the same concentration, shaking culturing at 28deg.C and 150rpm for 2-2.5h, determining OD600 between 0.5-0.8, adding inducer IPTG (purchased from AMRESCO) with final concentration of 0.5mM, continuing shaking culturing at 28deg.C and 150rpm for 4.5h, centrifuging at 6000rpm for 5min, and collecting bacterial precipitate; the bacterial pellet was resuspended in 50ml 20mM PBS (pH 7.4), centrifuged at 6000rpm for 15min and the pellet was collected.
4.2 high pressure crushing:
bacterial pellet was resuspended in 30ml 20mM PBS (pH 7.4) and broken at low temperature and high pressure, parameters set: the pressure value is 910-980bar, the cycle is 3 times, the temperature is set to 4 ℃, and the actual operation temperature is not higher than 13 ℃. After the completion of the crushing, the crushed product was centrifuged at 9000rpm at low temperature for 30min, and the supernatant was collected.
4.3 protein purification:
taking a nickel ion affinity chromatographic column: IMAC Bestarose-FF (purchased from Boglaung) was subjected to affinity chromatography and the column was equilibrated with 10 volumes of ultrapure water; then, a 10-time column volume balancing buffer (20 mM Tris-HCl pH 8.0) is used for balancing the chromatographic column, and then a protein sample is added; after loading, unbound protein was washed away with 10 column volumes of equilibration buffer (20 mM Tris-HCl pH 8.0); the mixed protein was washed with 10 column volumes of wash buffer (20 mM Tris-HCl-50mM IM pH 8.0), and finally the target protein was eluted with elution buffer (20 mM Tris-HCl-250mM IM pH 8.0).
The collected protein samples were placed in dialysis bags and placed in beakers containing dialysate (20 mM Tris-HCl pH 8.0) and dialyzed overnight at 4 ℃. And (3) carrying out SDS-PAGE on the purified protein sample to detect the purification condition, and simultaneously detecting whether a polymer exists or not by using a steric hindrance high performance liquid chromatography (SEC-HPLC), wherein the analysis result of the purity is shown in figure 5, the target size of the protein is about 35kD, the purity is more than or equal to 95%, and the protein is in a single form and has no aggregate.
Control antigens were also prepared according to the same embodiment.
Example 5: application of recombinant typhoid bacillus flagella antigen in gold-labeled chromatographic typhoid bacillus antibody detection reagent
5.1 colloidal gold-marking
1mL of colloid Jin Fang is taken in a glass cup, and a proper amount of 0.2MK is added by stirring 2 CO 3 Adjusting pH to 7.6, and stirring for 1min; adding 20 mug recombinant typhoid bacillus flagella antigen, stirring for 1min; 100 mu L of 1% PEG20000 is added and stirred for 1min; centrifuge at 5000g for 10 min, discard supernatant, and resuspend the pellet with 100ul of colloid Jin Xishi (20mM PB,150mM NaCl,0.1%PEG20000,0.1%TritonX-100,2% Sucrose,0.01% NaN 3); fully and uniformly mixing and preserving at 4 ℃. The gold-labeled compound is diluted by colloid Jin Xishi liquid 10 times and then uniformly coated on glass fiber, and is dried at 37 ℃ to prepare the gold-labeled pad.
5.2 nitrocellulose film (NC film) coating
The formula of the coating diluent comprises the following components: 0.01M PB, pH7.4, trehalose with mass percentage concentration of 0.05%, coating a detection line and a quality control line on the NC film in sequence from bottom to top, and baking at 37 ℃ for 30min.
5.3 Assembly
And assembling the immobilized NC film with a sample pad, an S pad, a gold Mark pad, a PVC bottom plate, absorbent paper and Mark paste, and cutting into test strips with the thickness of 3-4 mm by a slitter for later use.
5.4 sensitivity detection
The prepared typhoid bacillus antibody detection test strip is used for detecting the typhoid bacillus positive quality control product, and the detection result is shown in figure 6. The result shows that under the optimal labeling concentration, the color development condition rH (which means recombinant typhoid bacillus flagellin antigen pET28 a-rH) is obviously higher than that of a control (the sequence is 145-418aa, only the C end has a 6 x His tag, no other additional amino acid) and shows that the rH sensitivity is obviously higher than that of the control, and the analysis of the difference between the control and rH amino acid sequences shows that the rH N and the C end additional amino acid sequences are helpful for labeling gold particles and the sensitivity is improved.
5.6 sample detection
The prepared typhoid bacillus antibody detection test strip is used for detecting 5 cases of typhoid bacillus positive serum and 10 cases of typhoid bacillus negative serum, and the results are shown in table 1: the recombinant typhoid bacillus flagella antigen can be used as a marking raw material to detect positive samples in a test strip, and has high sensitivity and deep color development; the positive samples can be detected by the control antigen 1, but the sensitivity is low and the color development is weak; and the control antigens 2 and 3 show color difference and have the condition of missing detection. From the result of detecting a negative sample, rH and the control antigen 1 have no false positive, and the specificity is excellent; the control antigens 2 and 3 all have different degrees of false positive phenomena and have poor specificity.
Table 1: sample detection
It will be understood that equivalents and modifications will occur to those skilled in the art in light of the present teachings and concepts, and all such modifications and substitutions are intended to be included within the scope of the present invention as defined in the accompanying claims.
Claims (10)
1. A recombinant typhoid bacillus flagella antigen is characterized in that the amino acid sequence is shown as SEQ ID NO. 4.
2. The recombinant typhoid bacillus flagella antigen according to claim 1, wherein the corresponding coding nucleotide sequence of the amino acid sequence shown as SEQ ID NO.4 is shown as SEQ ID NO. 3.
3. A recombinant typhoid bacillus flagellin antigen according to claim 1, wherein the amino acid sequence of SEQ ID No.4 comprises an expression vector and a fragment of the typhoid bacillus flagellin (H) 145-418aa sequence.
4. A recombinant typhoid bacillus flagellin antigen according to claim 3, wherein the fragment of the typhoid bacillus flagellin (H) 145-418aa sequence is shown in SEQ ID No. 1.
5. A recombinant expression vector is characterized by being recombined by an expression vector and the codon optimization nucleotide sequence of the recombinant typhoid bacillus flagella antigen disclosed in claim 4, wherein the codon optimization nucleotide sequence is shown as SEQ ID NO. 2.
6. A recombinant engineering bacterium, which is prepared by transforming the recombinant expression vector according to claim 5 into a host strain.
7. A method of preparing a recombinant typhoid bacillus flagella antigen according to claim 1, comprising the steps of:
s1, selecting a typhoid bacillus flagellin 145-418aa fragment through sequence homology and antigenicity analysis, obtaining a nucleotide sequence for translating the amino acid through codon optimization, introducing BamHI and EcoRI enzyme cutting sites into the 5 'end and the 3' end, and connecting a target gene shown in SEQ ID NO.2 into a prokaryotic expression vector pET28a through gene synthesis to successfully obtain a recombinant vector pET28a-rH;
s2, transforming the recombinant expression vector into host bacteria, and screening to obtain recombinant engineering bacteria;
s3, culturing and inducing the recombinant engineering bacteria to express, and separating and purifying the product to obtain the recombinant typhoid bacillus flagella antigen.
8. Use of the recombinant typhoid bacillus flagella antigen according to claim 1 in an immunodiagnostic kit.
9. A kit for detecting antibodies to typhoid bacillus, characterized in that the recombinant typhoid bacillus flagella antigen as claimed in claim 1 is used as a diagnosis antigen.
10. The typhoid bacillus antibody detection kit according to claim 9, wherein the detection kit is a colloidal gold detection kit.
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