CN114163507B - Recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease and kit containing antigen - Google Patents
Recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease and kit containing antigen Download PDFInfo
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Abstract
The invention provides a recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease and a kit containing the antigen, belonging to the technical field of immunology, wherein the recombinant antigen protein comprises an amino acid sequence shown in a sequence table SEQ ID NO. 1. The kit comprises recombinant antigen protein rP44-60, a sample pad, a binding pad, an absorption pad, a nitrocellulose membrane, a colloidal gold probe, mouse IgG and sheep anti-mouse IgG. The recombinant antigen protein has high sensitivity and strong specificity, can rapidly detect antibodies in the serum infected by the phagostimulant intangible body, improves the problem that the antibodies are difficult to capture due to antigen variation of HGA, overcomes the defect of missed detection of the existing antigen, and effectively reduces the missed diagnosis rate; the preparation method reduces the cross reactivity caused by irrelevant sequences, and has high expression quantity and stability of recombinant antigen protein; the kit has the characteristics of rapidness, sensitivity, high specificity, no limitation of experimental conditions and the like, and has low requirements on detection conditions.
Description
Technical Field
The invention relates to the technical field of immunology, in particular to a recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease and a kit containing the antigen.
Background
Human granulocyte-free disease (Human granulocytic anaplasmosis, HGA) is a tick-borne disease which is caused by the invasion of human peripheral blood neutrophils by phagocyte-free cells (Anaplasma phagocytophilum) and is mainly clinically manifested by febrile leukopenia, thrombocytopenia and impairment of multiple organ functions. The disease is transmitted by a parasitic bacterium that is parasitic in cells, mainly by tick bites. The incubation period of the disease is 1-2 weeks, most of the diseases are caused acutely, and the fever can reach more than 40 ℃ after the diseases are continuously heated. The clinical manifestations of the disease are mainly general discomfort, hypodynamia, headache, muscle soreness, nausea, vomiting, anorexia, diarrhea and the like, possibly accompanied by multiple organ function damages of heart, liver, kidney and the like, and death can be caused when serious.
At present, HGA is a new zoonotic infectious disease with high international attention, and has become public health problem. Research has found that in most countries in the united states and europe, the rate of eosinophil intangible infection is often relatively high in areas where ticks are present. In addition, some mammals may also be storage hosts that are anaplasma phagocytes, such as white foot mice, white tail deer, red deer in europe, cattle, goats, etc., found in the united states. The existence of HGA is proved for the first time in the Anhui province in 2008 in China. The ministry of health, guidelines, states that phagocyte anaplasma was detected in full ditch hard ticks in the areas of Heilongjiang, inner Mongolia and Xinjiang, etc.
Early detection of phagocyte anaplast is important in controlling its spread, and therefore its differential detection is particularly important. HGA is not subject to any specific clinical symptoms, and therefore is usually detected by a serological detection method. The serological detection method is to detect specific antibodies against the pathogenic bacteria in the serum of an infected object by taking specific species proteins from the anaplasma phagocytes as detection antigens, so as to judge whether the infected object is infected with the anaplasma phagocytes, and the principle of specific resistance is the key point of the detection of the quality. Currently, the detection antigen used in serological detection methods is the phagocyte anaplasma outer membrane protein P44. However, because of the presence of the repeated sequences of the P44 multigene family in the genome of the intangible body, the same genes are easily exchanged, so that the organism has different P44 outer membrane proteins to generate antigen variation, for example, when the antigen of the cells is cultured by using the sensing HL60 and THP-1 respectively, the sensing HL60 cells can express a plurality of P44 clone mRNA, and the sensing THP-1 only expresses 2P 44 clone mRNA (P44-47E and P44-60). It can be seen that different P44 outer membrane proteins are produced on the surface of the anaplasma phagocytes in different tissue culture cells. The antigen variation can increase the detection difficulty and has the defect of omission, so that the detection sensitivity of the existing antigen can only reach 80-90%, and the detection sensitivity needs to be improved.
In order to solve the problems of difficult antibody detection, missed detection defect, lack of high-sensitivity high-specificity serum detection reagent, difficult popularization of detection in a basic layer and the like in HGA serum detection, a recombinant antigen protein is provided to strengthen the optimization of a granulocytopenia detection method, and has very important practical significance in the fields of medicine, public health and animal husbandry.
Disclosure of Invention
The invention provides a recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease, which is used for solving the problems of difficult antibody detection, missed detection defect and lack of high-sensitivity high-specificity serum detection reagent in HGA serum detection. The recombinant antigen protein provided by the invention has high sensitivity and strong specificity, can rapidly detect specific antibodies in the serum infected by the anaplast of phagocytic cells, improves the problem that the antibodies are difficult to capture due to antigen variation of HGA, overcomes the defect of missed detection of the existing antigen, effectively reduces the missed diagnosis rate, is beneficial to strengthening the optimization of the detection method of the granulocytoanaplasmosis, and has very important practical significance in the fields of medicine, public health and animal husbandry.
In a first aspect, the present invention provides a recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease, comprising: the amino acid sequence shown in SEQ ID NO.1 of the sequence table.
Further, the recombinant antigen protein rP44-60 consists of an amino acid sequence shown in the sequence table SEQ ID NO. 1.
Further provided is the use of the recombinant antigen protein rP44-60 in the preparation of a kit for detecting anaplasma phagocytosis antibodies.
The recombinant antigen protein provided by the invention maintains antigen reactivity in the preparation process, reduces cross reactivity caused by a similarity sequence in p44 polygene families, has better specificity and sensitivity, is easy to combine with phagocyte anaplasma antibodies in patient serum, can be used as an antigen for detecting granulocytoplasmatic diseases, is used for rapidly detecting the granulocytoplasmatic diseases, and provides scientific basis for rapid and simple detection of the diseases.
In a second aspect, the invention also provides a preparation method of the recombinant antigen protein rP44-60 for detecting the granulocytoanaplasmosis, which reduces cross reactivity caused by irrelevant sequences as much as possible, has high antigen protein expression, and overcomes the defects of low expression level and difficult preparation of the p44 whole genome antigen.
Specifically, the preparation method of the recombinant antigen protein rP44-60 comprises the following steps:
RT-PCR amplification, purification and recovery of p44 gene to obtain recovered product;
connecting the recovered product with a pCR2.1 vector, then converting the recovered product into competent cells DH5 alpha for induced expression, and sequencing and screening a target gene containing rP44-60 sequences;
ligating the target gene to a vector pTD1, and then converting the target gene into competent cells DH5 alpha again for cloning culture, and then extracting, identifying and collecting positive recombinant plasmids; the method comprises the steps of,
and after sequencing the positive recombinant plasmid, synthesizing target protein and purifying to obtain recombinant antigen protein rP44-60.
According to the preparation method provided by the technical scheme, on the premise of retaining the antigen reactivity to the greatest extent, the cross reactivity caused by irrelevant sequences is reduced as much as possible, so that the expression quantity and stability of the recombinant antigen protein are improved, and the defects that the p44 whole genome antigen expression quantity is low and the preparation is difficult are overcome; the prepared recombinant antigen protein rP44-60 is used as an antigen, can effectively reduce the detection cost and is easy to standardize.
In a specific embodiment, in the RT-PCR amplification step, the PCR amplification is performed in two rounds, and the conditions for the two rounds of PCR amplification are the same, specifically as follows: pre-denaturation at 94 ℃ for 30s-5min, denaturation at 94 ℃ for 30-60s, annealing at 55-60 ℃ for 30-60s, extension at 72 ℃ for 1-2min, and reaction for 45 cycles.
Further, in the RT-PCR amplification step, the upstream primer of round 1 amplification is 5'-GCTAAGGAGTTAGCTTATGA-3', and the downstream primer is 5'-CAATAGT (C/T) TTAGCTAGTAACC-3'; the upstream primer of round 2 amplification was 5'-CTGCTCT (T/G) GCCAA (A/G) ACCTC-3', and the downstream primer was 5'-AGAAGATCATAACAAGCATTG-3'.
In a specific embodiment, the medium used in the step of inducing expression is: plate medium containing 1mM IPTG and 1mM X-gal; the culture medium used in the cloning culture step is as follows: selection medium containing 100. Mu.g/mL of Ampicillin antibiotic.
Further, the plate culture medium and the selection culture medium are LB culture medium containing 0.002-0.010 g/L-isoleucine tert-butyl ester hydrochloride and 0.001-0.015g/L compound sodium nitrophenolate. The synergistic effect of L-isoleucine tert-butyl ester hydrochloride and sodium nitrophenolate is utilized to protect and promote the flow of cell protoplasm, so as to effectively improve the activity of cell, accelerate the growth and propagation of cell, and help to improve the biomass of thallus, and the amino group and other structures in molecular structure are possibly utilized to strengthen and promote the correct folding of protein to obtain recombinant protein, so as to prevent the protein from gathering to form inclusion bodies, so that the recombinant protein has sufficient space conformation, the bioactivity and stability of the recombinant protein are ensured, and the expression quantity of the recombinant protein is improved.
Specifically, the raw materials and the contents of the plate medium and/or the selection medium are as follows: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, 0.002-0.010g/L of L-isoleucine tert-butyl ester hydrochloride and 0.001-0.015g/L of compound sodium nitrophenolate. Further, the plate culture medium is a solid culture medium, and the selective culture medium is a liquid culture medium. The culture medium can improve the success rate and the yield of the recombinant protein expressed by escherichia coli in cytoplasm, improve the expression quantity and the stability of the recombinant antigen protein, improve the phenomena of small bacterial growth quantity, low target protein yield, poor stability and the like existing in the common LB culture medium, and not only can meet the subsequent research requirements, but also can meet the large-scale production requirements.
More specifically, the preparation method of the recombinant antigen protein rP44-60 comprises the following steps:
1) The original proteome is synthesized by using bioinformatics technology according to the existing p44 multi-genome sequence as a basic template, and then the original proteome is sequenced to determine that the original proteome contains rP44-60 target gene sequences.
2) Reverse transcription to cDNA was performed using RNA extraction kit and RNA extracted from the infected THP-1 cultured cells as a template according to the reverse transcription kit. Designing a primer according to the target gene sequence, then carrying out RT-PCR amplification, and recovering the amplified product by agarose gel electrophoresis to obtain a recovered product. Wherein, the reverse transcription conditions are: 37-42 ℃ for 15-45min; denaturation at 85℃for 5-60s.
3) The recovered product was ligated to pCR2.1 vector, and then the ligation product was transformed into competent cells DH 5. Alpha. For protein expression, and induced and cultured on a plate medium containing 1mM IPTG and 1mM X-gal at 37.+ -. 2 ℃ and at 200-500rpm for 3-5 hours. Screening and separating by a blue-white spot method, and picking white colonies for subculture. The cells were collected by centrifugation, and after the cells were sonicated, the gene fragment was extracted, and the inserted fragment was confirmed by using restriction enzyme EcoRI, followed by sequencing. Sequence analysis was performed using Blast search and MEGA7.0 software, and a target gene containing rP44-60 sequences was collected in a high expression level.
Wherein, the ultrasonic crushing conditions are as follows: the power is 500-600w, the ultrasonic time is 2s, the interval is 5s, and the total cycle is 100 times. Intermittent crushing is adopted, so that the situation of protein loss caused by too severe crushing can be avoided.
4) The target gene with correct sequencing is connected to a plasmid vector pTD1 containing a T7 promoter and transformed into competent cells DH5 alpha prepared by a calcium chloride method to obtain a transformant. The transformed bacteria are plated on a selection medium containing 100 mug/mL of Ampicillin antibiotics, and then are subjected to cloning culture for 12-16 hours at 37+/-2 ℃ until the OD value of the bacteria liquid is not lower than 0.5. The plasmid of the cloned colony is then extracted and collected, and the supernatant and precipitate are separated by ultrasonic washing.
5) The collected plasmid is subjected to ultrasonic washing and purification to obtain rP44-60-pTD1 plasmid DNA, then the rP44-60-pTD1 plasmid DNA is subjected to PCR amplification and enzyme digestion identification, restriction enzyme EcoRv is adopted for enzyme digestion, and then positive recombinant plasmid is sequenced.
6) After sequencing, preparing and synthesizing mRNA in large quantity by utilizing the positive recombinant plasmid, synthesizing target protein, and purifying to obtain recombinant antigen protein rP44-60.
In a third aspect, the present invention also provides a kit containing recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease, the kit being used for detecting phagocytic intangible antibodies, comprising the recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease.
The kit has the characteristics of rapidness, sensitivity, high specificity, no limitation of experimental conditions and the like, has low requirements on detection conditions, and is suitable for detecting the granulocytoplasmatic disease in clinical rural areas, particularly with limited conditions.
In a specific embodiment, the above kit further comprises: sample pad, conjugate pad, absorbent pad, nitrocellulose membrane, colloidal gold probe, mouse IgG and sheep anti-mouse IgG. Compared with an immunofluorescence method, the kit can greatly improve the detection sensitivity, has higher specificity, has the characteristics of rapidness and simplicity, and is suitable for primary hospitals.
Preferably, the sample pad and the bonding pad are made of glass fibers, and the absorption pad is made of water-absorbing filter paper.
The recombinant antigen protein rP44-60 for detecting the granulocytoplasmatic disease and the preparation method thereof provided by the invention have the beneficial effects that the recombinant antigen protein rP44-60 is prepared into a kit for detecting the phagocytic anaplasmatic antibody, and the kit is further used for the technical means of detecting the granulocytoplasmatic disease, so that the following beneficial effects are realized:
1) The recombinant antigen protein provided by the invention is convenient and quick to use as an antigen, has high sensitivity and strong specificity, has no cross reaction with other pathogens, can solve the problem that the antibody is difficult to capture due to antigen variation of HGA, overcomes the defect of missed detection of the existing detection antigen, improves the detection and detection sensitivity of the granulocytoanaplasmosis, effectively reduces the missed diagnosis rate, is favorable for rapidly and accurately detecting the occurrence of the granulocytoanaplasmosis clinically, and has higher popularization and application value and wide application prospect.
2) The preparation method of the invention reduces cross reactivity caused by irrelevant sequences as much as possible on the premise of retaining antigen reactivity to the greatest extent, has high expression quantity and stability of recombinant antigen protein, and overcomes the defects of low expression quantity and difficult preparation of p44 whole genome antigen. The prepared recombinant antigen protein rP44-60 can be used as an antigen, can effectively reduce the detection cost and is easy to standardize.
3) The double-antigen sandwich colloidal gold immunochromatography adopted by the kit has the characteristics of rapidness, simplicity, convenience, low cost, no pollution and no need of training, is more suitable for on-site detection compared with the traditional methods such as IFA, ELISA and the like, has the advantages of short color development time, no need of expensive instruments and the like, has low requirements on detection conditions, is suitable for detecting granulocytoplasms in rural areas with clinical and especially limited conditions, and has wide market prospect and application value.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the detection result of WB technology of the antigenicity of recombinant antigen protein rP 44-60;
FIG. 2 shows the results of assays performed on serum to be tested using different kits.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. 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 also within the scope of the invention.
Further, the target protein is purified to obtain recombinant antigen protein, and the purification modes can be various, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography and the like. In some embodiments of the invention, the recombinant antigen protein is preferably purified using nickel affinity chromatography, and the recombinant antigen protein of interest is eluted with an Elution Buffer (50mM Tris,0.2M NaCl,0.5M Imidazole,pH8.0), which can be identified by SDS-PAGE. The purified recombinant antigen protein has higher purity.
The kit is prepared by adopting a double-antigen sandwich colloidal gold immunochromatography method, wherein the preparation method of the kit by adopting the colloidal gold immunochromatography detection strip is as follows:
1) And (3) preparing a colloidal gold probe liquid by taking a colloidal gold probe as a tracer marker, and then preparing a recombinant antigen protein rP44-60 colloidal gold complex and a mouse IgG colloidal gold complex respectively.
2) Mixing the recombinant antigen protein rP44-60 colloidal gold complex and the mouse IgG colloidal gold complex, re-suspending with 0.1mmol/L Tris solution containing 5% sucrose and 5% BSA, adjusting the re-suspension to OD530 to 2, soaking the binding pad, and drying at 37 ℃ for 2-4h to obtain the gold-labeled pad.
3) Marking recombinant antigen protein rP44-60 point membrane liquid as a detection line, marking sheep anti-mouse IgG point membrane liquid as a quality control line on a nitrocellulose membrane, and drying at 37 ℃ overnight; the protein concentration in the recombinant antigen protein rP44-60 point membrane solution is 4mg/mL, and the streaking parameter is 0.1 mu L/mm; the concentration of the antibody in the goat anti-mouse IgG spot membrane solution is 5mg/mL, and the streaking parameter is 0.1 mu L/mm. The dilution liquid for the spot film liquid is prepared as follows: 50mM Tris solution containing 2% sucrose, pH 8.5. The scribing distance between the detection line and the quality control line is 7+/-2 mm.
4) And sequentially superposing or assembling the absorption pad, the nitrocellulose membrane, the gold-labeled pad and the sample pad on a bottom plate, then cutting the chromatographic strip, and drying and keeping the chromatographic strip at room temperature in a dark place, wherein the width of the chromatographic strip is 3-5 mm/strip, thus obtaining the colloidal gold immunochromatographic strip for the kit.
In a specific embodiment, the method of using the above kit is as follows: taking out the colloidal gold immunochromatography detection strip, then dripping 70-100 mu L of the liquid to be detected onto a sample pad, standing for 20min at room temperature, observing, and judging the detection result. The decision criteria are as follows:
1) When the color bands appear on the detection line and the quality control line, the detection line and the quality control line are positive;
2) Only the quality control line is provided with a color band, and no color band appears in the test area, and the test area is negative;
3) When the quality control line does not develop color, the detection strip is damaged or fails, and whether the detection line has color bands or not, the detection strip should be replaced by a new detection strip for retesting.
In some embodiments of the invention, the kit is used for detecting granulocytoplasms, and when the detection result is positive, the kit represents that the biological sample contains the antibody of the anaplasma phagocytes, which means that the test organism has the anaplasma phagocytes or has been infected by the anaplasma phagocytes.
In some embodiments, the kits of the invention can further comprise a set of instructions that instruct, for example, how to use the kits of the invention to detect antibodies to anaplasma antigens in a subject.
It should be noted that the recombinant antigen protein rP44-60 of the present invention can be used as an antigen for detecting granulocytoplasmatic disease, and can detect specific antibodies in animal serum by various serological methods to detect whether the animal is infected with phagocytic anaplasma. Serological methods include, but are not limited to ELISA, DB, WB, IFA, animal serum including, but not limited to, humans, dogs, cats, horses, and the like. Further, the test animal sample is not limited to serum, but can be blood, plasma, cerebrospinal fluid, tissue extract, urine, or saliva samples.
The system, operation, steps and the like which are not described in detail in the invention refer to a third edition of molecular cloning experiment guidelines, such as the use of a kit and the like, which are operated according to the specification of the corresponding kit; unless otherwise indicated, the instruments and reagent materials used in the following examples are conventional instruments and reagent materials used by those skilled in the art, and are not described herein.
The present invention will be described in further detail with reference to examples.
Example 1:
a preparation method of recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease comprises the following steps:
1) The original proteome is synthesized by using bioinformatics technology and taking the sequence of the existing p44 multiple genome as a basic template, and then the original proteome is subjected to Gene Script sequencing to determine that the original proteome contains rP44-60 target Gene sequences.
2) RNA extracted from the cells cultured in the THP-1-sensitive culture was used as a template using the RNA extraction kit (QIAGEN), and reverse transcribed into cDNA according to the instructions of the reverse transcription kit. Primers were designed according to the target gene sequence, then RT-PCR amplification was performed, and the amplified product (TaKaRa's DNA recovery and purification kit) was recovered using agarose gel electrophoresis to obtain a recovered product. The reverse transcription conditions are as follows: 37 ℃ for 15min; denaturation at 85℃for 5s.
In the RT-PCR amplification step, the PCR amplification is carried out for two rounds, and the conditions of the two rounds of PCR amplification are the same, specifically as follows: pre-denaturation at 94℃for 30s, annealing at 55℃for 60s, extension at 72℃for 1min, 45 cycles of reaction.
Wherein, the upstream primer of the 1 st round of amplification is 5'-GCTAAGGAGTTAGCTTATGA-3', and the downstream primer is 5'-CAATAGT (C/T) TTAGCTAGTAACC-3'. The upstream primer of round 2 amplification was 5'-CTGCTCT (T/G) GCCAA (A/G) ACCTC-3', and the downstream primer was 5'-AGAAGATCATAACAAGCATTG-3'.
3) The recovered product was ligated to pcr2.1 vector (Invitrogen), and the ligation product was then transformed into competent cells DH5 a (TOYOBO) for protein expression and induced and cultured for 3.5h at 37 ℃ and 300rpm on plate medium containing 1mM IPTG and 1mM X-gal. Screening and separating by a blue-white spot method, and picking white colonies for subculture. The cells were collected by centrifugation, and after the cells were sonicated, the gene fragment was extracted, and the inserted fragment was confirmed by using restriction enzyme EcoRI, followed by sequencing. Sequence analysis was performed using Blast search and MEGA7.0 software, and a target gene containing rP44-60 sequences was collected in a high expression level. The ultrasonic crushing conditions are as follows: the power is 500w, the ultrasonic time is 2s, the interval is 5s, and the total cycle is 100 times.
4) The target gene with correct sequencing was ligated to a plasmid vector pTD1 containing a T7 promoter using an In-Fusion kit and transformed into competent cells DH 5. Alpha. Prepared by the calcium chloride method to give a transformant. The transformed bacteria are plated on a selection medium containing 100 mug/mL of Ampicillin antibiotics, and then are subjected to cloning culture for 16 hours at 37 ℃ until the OD value of the bacterial liquid is not lower than 0.5. The plasmid of the cloned colony is then extracted and collected, and the supernatant and precipitate are separated by ultrasonic washing.
5) The collected plasmid is subjected to ultrasonic washing and purification to obtain rP44-60-pTD1 plasmid DNA, then the rP44-60-pTD1 plasmid DNA is subjected to PCR amplification and enzyme digestion identification, restriction enzyme EcoRv is adopted for enzyme digestion, and then positive recombinant plasmid is sent to Greiner Japan for sequencing.
6) After sequencing, mRNA is prepared and synthesized in a large scale by using a T7 riboMAXTM large scale RNA production system (Promega) in a test tube, then a target protein is synthesized by using a protein synthesis kit Transdirect insect cell (SHIMADZU), and finally the target protein is purified by adopting an affinity chromatography to obtain recombinant antigen protein rP44-60.
The raw materials and the contents of the plate medium and the selection medium used in the above steps 3) and 4) are as follows: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride, 0.005g/L of L-isoleucine tert-butyl ester hydrochloride and 0.01g/L of compound sodium nitrophenolate.
Example 2:
the embodiment provides a kit based on double-antigen sandwich colloidal gold immunochromatography, which comprises recombinant antigen protein rP44-60 for detecting granulocytoanaplasmosis.
Specifically, the preparation method of the colloidal gold immunochromatographic assay strip for the kit comprises the following steps:
1) And (3) preparing a colloidal gold probe liquid by taking a colloidal gold probe as a tracer marker, and then preparing a recombinant antigen protein rP44-60 colloidal gold complex and a mouse IgG colloidal gold complex respectively.
2) The recombinant antigen protein rP44-60 colloidal gold complex and the mouse IgG colloidal gold complex are mixed, resuspended in 0.1mmol/L Tris solution containing 5% sucrose and 5% BSA, and then the resuspension is adjusted until the OD530 reaches 2, and the binding pad is soaked and dried for 4 hours at 37 ℃ to prepare the gold-labeled pad.
3) Marking recombinant antigen protein rP44-60 point membrane liquid as a detection line, marking sheep anti-mouse IgG point membrane liquid as a quality control line on a nitrocellulose membrane, and drying at 37 ℃ overnight; the protein concentration in the recombinant antigen protein rP44-60 point membrane solution is 4mg/mL, and the streaking parameter is 0.1 mu L/mm; the concentration of the antibody in the goat anti-mouse IgG spot membrane solution is 5mg/mL, and the streaking parameter is 0.1 mu L/mm. The dilution liquid for the spot film liquid is prepared as follows: 50mM Tris solution containing 2% sucrose, pH 8.5. The scribing distance between the detection line and the quality control line is 7mm.
4) And sequentially superposing or assembling the absorption pad, the nitrocellulose membrane, the gold-labeled pad and the sample pad on a bottom plate, then cutting the chromatographic strip, drying at room temperature and keeping the chromatographic strip away from light, thus obtaining the colloidal gold immunochromatography detection strip for the kit.
The above mouse IgG and sheep anti-mouse IgG were purchased from beijing boerci technologies.
Comparative example 1:
the preparation method of recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease is different from example 1 only in that:
the raw materials and the contents of the plate medium used in step 3) and step 4) are as follows: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 0.005g/L of L-isoleucine tert-butyl ester hydrochloride, and no compound sodium nitrophenolate is added.
Comparative example 2:
the preparation method of recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease is different from example 1 only in that:
the raw materials and the contents of the plate medium used in step 3) and step 4) are as follows: 10g/L of tryptone, 5g/L of yeast extract, 10g/L of sodium chloride and 0.01g/L of compound sodium nitrophenolate, and L-isoleucine tert-butyl ester hydrochloride is not added.
Comparative example 3:
the preparation method of recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease is different from example 1 only in that:
the flat-plate culture medium used in the step 3) and the step 4) is a conventional LB culture medium, and the raw materials and the content thereof are as follows: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride.
Experimental example 1:
antigenicity detection
The experimental method comprises the following steps: the recombinant antigen protein rP44-60 prepared in example 1 was used as an antigen, and the antigenicity of the serum of an animal infected with a phagostimulant and a positive patient was detected by the WB technique, and the feasibility of the antigen was evaluated. In animals, 3 parts of mouse positive serum, 3 parts of sheep positive serum and 3 parts of positive patient serum. The secondary antibody was a coat antibody-Human IgG Alkaline phosphatase (Invitrogen). Firstly, recombinant antigen protein rP44-60 is loaded on an SDA-PAGE gel, the protein is transferred to a nitrocellulose membrane, the membrane is cut into strips, the strips are closed for 30min, incubated with primary anti-positive serum (1:2000), PBS is used for 3 times, secondary anti-positive serum (1:5000) is used for 4 times, and development is carried out. The results are shown in FIG. 1.
FIG. 1 shows the detection result of the WB technology of the antigenicity of recombinant antigen protein rP44-60. Wherein, 1-3 is positive serum of mice, 4-6 is positive serum of sheep, and 7-9 is positive serum of patients. The result shows that 9 parts of positive serum can react with recombinant antigen protein rP44-60, and the recombinant antigen protein rP44-60 and the anaplasma phagocytophilic positive serum both show stronger positive reaction, so that the recombinant antigen protein rP44-60 has stronger antigenicity and can be used as an antigen.
Experimental example 2:
kit detection test
The experimental method comprises the following steps: the kit and the test strip prepared in example 2 were used as sample 1, and the recombinant protein P44-1 (amino acid sequence shown in SEQ ID NO. 2) conventionally used in the prior art was used as a control, and the kit and the test strip prepared in example 2 were used as sample 2. Providing 20 parts of phagocyte anaplasma infection positive dog serum and 15 parts of negative dog serum; 30 parts of phagocyte intangible body infected positive human serum and 30 parts of negative human serum. The total of 95 serum to be tested is detected by using two samples respectively, and the detection results are counted and compared. And (5) counting the sensitivity and the specificity of the detection, and calculating the overall coincidence rate. Each experimental group was set with 3 replicates and averaged. The results are shown in FIG. 2.
FIG. 2 shows the results of assays performed on serum to be tested using different kits. The result shows that 50 positive parts are detected in 50 positive serum by using a sample 1 kit of recombinant antigen protein rP44-60, and 0 part is missed; false positive 1 in 45 negative serum; the calculation results are that: the sensitivity is 100%, the specificity is 97.8%, and the overall coincidence rate is 98.9%. Using a sample 2 kit of recombinant protein P44-1, detecting 49 positive parts in 50 positive serum, and missing 1 part; false positive 3 parts appear in 45 parts of negative serum; the calculation results are that: the sensitivity is 98.0%, the specificity is 93.3%, and the overall coincidence rate is 95.8%.
The recombinant antigen protein rP44-60 and the kit thereof have high sensitivity and specificity when detecting the anaplasma phagocytosis antibody, can be used as raw materials for preparing the kit/detection strip for detecting the anaplasma phagocytosis antibody, can be widely applied to clinical detection, and can effectively reduce the problems of antigen variation, omission and the like in the serum detection of HGA because of low homology of the two proteins and low possibility of cross reaction in serology.
Experimental example 3:
influence of different media on protein expression
The experimental method comprises the following steps: the plate media in example 1 and comparative examples 1 to 3 were taken as experimental samples, respectively. Preparing the transformed bacteria of step 4) of example 1 to a bacterial concentration of1×10 7 Seed liquid of each mL is coated on several plate culture medium samples according to the proportion of 5% by volume, the temperature during the culture is 37 ℃ and the rotating speed is 140rpm. The bacterial concentration was measured 8 a day in the morning during the culture period, and after the culture was completed by day 7, colonies were collected and crushed, and the ELISA method was provided to measure the expression level of each group protein. Each experimental group was set with 3 replicates and averaged. The results are shown in Table 1.
Table 1 shows the results of the effects of different culture media on the concentration of bacteria and the expression level of protein
| Example 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| Final bacterial concentration (individual/mL) | 1.9×10 8 | 0.3×10 8 | 6.8×10 7 | 7.6×10 7 |
| Highest concentration of bacteria (individual/mL) | 9.1×10 8 | 4.8×10 8 | 1.6×10 8 | 5.3×10 8 |
| Protein expression level (mg/L) | 71.73 | 62.49 | 57.37 | 56.64 |
The results showed that the experimental group using the culture medium of comparative examples 1 to 3 was lower in both the highest bacterial concentration and the final bacterial concentration during the culture to different extents than the experimental group using the culture medium of example 1; the protein expression amount after the end of the culture was also significantly higher in example 1 than in comparative examples 1 to 3. Comprehensive description shows that the culture medium components in the embodiment 1 synergistically improve the cell viability, accelerate the cell growth and propagation, help to improve the biomass of thalli, ensure the bioactivity and stability of recombinant proteins, and ensure that the expression quantity of the recombinant proteins is obviously improved.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solution of the present invention, and not limiting thereof; although the invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will appreciate that; the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Sequence listing
<110> river set college
<120> recombinant antigen protein rP44-60 for diagnosis of granulocytoplasmatic disease and kit containing the same
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Leu Ala Ala Asn Thr Asn Ala Glu Lys Lys Tyr Ala Val Glu Pro Ala
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Asn Gly Gly Thr Asp Gly Ser Thr Ser Gln Cys Ser Gly Leu Ser Asn
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Gly Ser Ala
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Claims (8)
1. A recombinant antigen protein rP44-60 for detecting granulocytoplasmatic disease, which is characterized in that: the recombinant antigen protein rP44-60 consists of an amino acid sequence shown in a sequence table SEQ ID NO. 1.
2. The recombinant antigen protein rP44-60 of claim 1 wherein: use of said recombinant antigen protein rP44-60 in the preparation of a kit for detecting anaplasma phagocytophil antibodies.
3. A method for preparing the recombinant antigen protein rP44-60 of claim 1 or 2, which is characterized in that: comprising the following steps:
p44RT ⁃ PCR amplification, purification and recovery of genes to obtain recovery products;
connecting the recovered product with a pCR2.1 vector, then converting the recovered product into competent cells DH5 alpha for induced expression, and sequencing and screening out a target gene containing rP44-60 sequences;
connecting the target gene to a vector pTD1, and converting the target gene into competent cells DH5 alpha again for cloning culture, and then extracting, identifying and collecting positive recombinant plasmids; the method comprises the steps of,
and after sequencing the positive recombinant plasmid, synthesizing target protein and purifying to obtain recombinant antigen protein rP44-60.
4. A method of preparation according to claim 3, characterized in that: in the RT ⁃ PCR amplification step, two rounds of PCR amplification are performed, and the conditions of the two rounds of PCR amplification are the same, specifically as follows: pre-denaturation at 94 ℃ for 30s-5min, denaturation at 94 ℃ for 30-60s, annealing at 55-60 ℃ for 30-60s, extension at 72 ℃ for 1-2min, and reaction for 45 cycles.
5. A method of preparation according to claim 3, characterized in that: the culture medium for the induced expression step is as follows: plate medium containing 1mM IPTG and 1mM X ⁃ gal; the culture medium for the clone culture step is as follows: selection medium containing 100. Mu.g/mL of Ampicillin antibiotic.
6. The method of manufacturing according to claim 5, wherein: the plate culture medium and the selection culture medium are LB culture medium containing 0.002-0.010 g/L-isoleucine tert-butyl ester hydrochloride and 0.001-0.015g/L compound sodium nitrophenolate.
7. A kit containing recombinant antigen protein rP44-60 for detecting granulocytoanaplasmosis, said kit being for detecting phagocytic anaplast antibodies, characterized in that: comprising the recombinant antigen protein rP44-60 for granulocytopenia detection according to claim 1.
8. The kit of claim 7, wherein: the kit further comprises: sample pad, binding pad, absorption pad, nitrocellulose membrane, colloidal gold probe, mouse IgG and sheep anti-mouse IgG; the sample pad and the bonding pad are made of glass fibers, and the absorption pad is made of water-absorbing filter paper.
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