CN110343161B - Binding protein composition for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a binding protein combination for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH, and a preparation method and application thereof. The combination of the binding proteins comprises a binding protein 1 and a binding protein 2 for detecting plasmodium falciparum HRP2, a binding protein 3 and a binding protein 4 for detecting plasmodium vivax LDH, and amino acid sequences of the binding proteins 1-4 are shown in SEQ ID NO. 5-8. The invention adopts phage display technology and takes plasmodium falciparum HRP2 and plasmodium vivax LDH as target antigens to select a binding protein combination with high panning affinity and good specificity from a phage display protein library. Recombinant expression and purification are carried out by utilizing a genetic engineering technology, the combined protein reagent replaces an antibody reagent used in malaria immunoassay, a colloidal gold chromatography rapid detection method is established, the storage stability of the obtained kit is higher than that of the existing clinical detection product, and the kit has the advantages of reliability, accuracy, safety, simplicity, convenience and stability.

Description

Binding protein composition for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH, and preparation method and application thereof

Technical Field

The invention relates to the technical field of phage display, in particular to a binding protein combination for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH, and a preparation method and application thereof.

Background

Malaria is one of the three most serious infectious diseases worldwide, about half of the population faces the risk of malaria, and in China, some regions still have malaria infection cases. Because the modes of malaria transmission, carrying, latent disease and the like are special, rapid diagnosis is very important in malaria detection.

Malaria detection technology has been developed to date, and clinical detection methods mainly include microscopic examination, immunodetection (immunochromatography, enzyme-linked immunosorbent assay) and PCR nucleic acid amplification tests. Wherein, microscopic examination and PCR nucleic acid amplification have the problems of low efficiency, expensive detection cost and the like; the immunoassay has the problems of difficult product storage, expensive raw materials and the like, and cannot be rapidly popularized and applied in developing countries with severe malaria propagation, such as southeast Asia and Africa.

Therefore, it is necessary to develop a new malaria detection method, which can rapidly and accurately detect malaria and reduce the detection difficulty and cost.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a combination of binding proteins for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH.

Another object of the present invention is to provide a combination of genes encoding the above combination of binding proteins.

Another object of the present invention is to provide a method for preparing the above-mentioned combination of binding proteins.

The invention also aims to provide the application of the combination of the binding proteins in preparing a reagent for detecting malaria.

Another object of the present invention is to provide a method for preparing a reagent for detecting malaria.

In order to achieve the purpose, the invention is realized by the following scheme:

a binding protein combination for detecting plasmodium falciparum HRP2 and plasmodium vivax LDH is composed of a binding protein 1 and a binding protein 2 for detecting plasmodium falciparum HRP2, a binding protein 3 and a binding protein 4 for detecting plasmodium vivax LDH, and amino acid sequences of the binding proteins 1-4 are shown as SEQ ID NO. 5-8.

A gene combination for coding the binding protein combination has a nucleotide sequence shown in SEQ ID NO. 1-4.

Wherein, the nucleotide sequence of the gene for coding the binding protein 1 is shown as SEQ ID NO. 1, the nucleotide sequence of the gene for coding the binding protein 2 is shown as SEQ ID NO. 2, the nucleotide sequence of the gene for coding the binding protein 3 is shown as SEQ ID NO. 3, and the nucleotide sequence of the gene for coding the binding protein 4 is shown as SEQ ID NO. 4.

The invention also claims a preparation method of the binding protein combination, which comprises the following steps: phage containing the above gene combination (consisting of nucleotide sequences encoding a combination of binding proteins specifically reactive with P.falciparum HRP2 and P.vivax LDH) was added to the culture broth inoculated with ER2738 E.coli, and cultured at 37 ℃ for 1 hour at 90 rpm; then coating on an LB agar culture plate, adding 100 mu g/mL carbenicillin, and culturing at room temperature overnight; the next day, the colonies were scraped into 5mL of 2TY medium and inoculated into 25mL of 2TY medium containing 100. mu.g/mL of carbenicillin to reach OD_600nmCulturing at 225 rpm at 37 deg.C for 1 hr (0.2), and culturing with 10 rpm⁹M13K07 helper phage infection of complexity; after incubation for 1 hour at 90 rpm, 25. mu.g/mL kanamycin was added, cells were incubated overnight at 25 ℃ at 170 rpm, and phage were incubated with 6% kanamycinPrecipitating with PEG8000 and 0.3M NaCl, and suspending in 1mL buffer solution containing 10mM Tris and 1mM EDTA, pH 8.0.

The invention also claims a preparation method of the binding protein combination, which comprises the following steps:

amplifying nucleotide sequences of the gene combination for coding the binding protein combination, and performing double enzyme digestion on the amplification product and pET11a plasmid by respectively adopting Nhel and Pstl restriction endonucleases; selecting clones, culturing in 5mL LB culture medium at 37 deg.C and 225 rpm overnight; the plasmid was transferred into E.coli BL21(DE3) cells by heat shock and cultured overnight at 37 ℃; the next day, the culture broth was added to 10mL of LB medium and cultured at 37 ℃ at 250 rpm to OD_600nm0.6, isopropyl β -D-1 thiogalactoside was added to a final concentration of 1 mM; culturing the cells for 6 hours again, centrifuging at the rotating speed of 3000g, and then suspending in 1mL of 1 XBugbuster protein extraction reagent; adding nuclease, mixing at room temperature, suspending for 20 min, heating to 50 deg.C, and centrifuging at 9400g for 20 min; the supernatant and Ni-NTA purification resin 50L mixing suspension 1 hours, pH7.4 containing 50mM PBS, 500mM NaCl, 20mM imidazole 30mL washing liquid 3 times, and then pH7.4 containing 50mM PBS, 500mM NaCl, 300mM imidazole 0.1mL elution, get the claim of claim 1 binding protein combination.

The invention also claims the application of the combination of the binding proteins in preparing a reagent for detecting malaria.

The invention also claims a preparation method of the reagent for detecting malaria, which comprises the following steps:

s1, preparing a colloidal gold bonding pad:

s11, adding 12 mu L of 0.1mol/L potassium carbonate solution into every 1mL of colloidal gold, shaking and uniformly mixing, adding binding protein 1 and binding protein 2 for detecting plasmodium falciparum HRP2 according to 16 mu g/mL, adding binding protein 3 and binding protein 4 for detecting plasmodium vivax LDH according to 12 mu g/mL, uniformly mixing, reacting for 40min, adding 1% PEG 2000020 mu L/mL of colloidal gold, uniformly mixing, sealing for 15min, centrifuging at 10000rpm for 15min, discarding supernatant, dissolving precipitate with 1000 mu L of gold-labeled re-solution, and standing at 4 ℃ for later use;

s12, preparing a mouse IgG antibody labeled colloidal gold conjugate, adding 12 mu L of 0.1mol/L potassium carbonate solution into 1mL of colloidal gold, shaking and uniformly mixing, adding the mouse IgG antibody according to 20 mu g/mL, uniformly mixing, reacting for 40min, adding 1% BSA (bovine serum albumin) and 25 mu L/mL of colloidal gold, uniformly mixing, sealing for 15min, centrifuging at 10000rpm for 15min, discarding supernatant, dissolving a precipitate with 1000 mu L of gold labeled re-solution, and standing at 4 ℃ for later use; the gold-labeled complex solution consists of the following components in concentration: 0.02mol/L Tris, 1% BSA, 0.1% Tween-20 and 20% sucrose;

s13, uniformly coating the colloidal gold conjugate obtained in the steps S11 and S12 on 15cm per 1mL after mixing²Drying the glass cellulose membrane at 37 ℃ for 22 hours, and sealing for later use;

s2, coating film scribing preparation:

s21, preparing a coating buffer solution: adding 5% sucrose into 10mmol/L PBS (pH 7.4);

s22, carrying out detection line marking on plasmodium falciparum HRP 2: diluting the binding protein 1 and the binding protein 2 to 1mg/mL, coating the diluted binding protein 1 and the binding protein 2 on a nitrocellulose membrane by using a film spraying machine according to the scribing concentration of 0.1 muL/mm, drying the nitrocellulose membrane at 37 ℃ overnight, and sealing and storing the nitrocellulose membrane for later use;

s23, scribing plasmodium vivax LDH detection lines: diluting the binding protein 3 and the binding protein 4 to 1mg/mL, coating the diluted binding protein 3 and the binding protein 4 on a nitrocellulose membrane by a film spraying machine according to the scribing concentration of 0.1 muL/mm, drying the nitrocellulose membrane at 37 ℃ overnight, and sealing and storing the nitrocellulose membrane for later use;

s24, scribing a quality control line C: scribing goat anti-mouse antibody with a scribing concentration of 1.5mg/mL according to a scribing concentration of 0.1 muL/mm, scribing and coating on a nitrocellulose membrane by using a film spraying machine, drying at 37 ℃ overnight, and sealing and storing for later use;

scribing the three reagents of the steps S22-S24 on the same nitrocellulose membrane, and sequentially starting from the end of water-absorbing paper to form a C line, a T2 line (Plasmodium vivax LDH detection line) and a T1 line (Plasmodium falciparum HRP2 detection line);

s3, preparing a sample pad:

s31, preparing a sample pad treating fluid: 1% PVP and 0.5% BSA are calculated and weighed to be dissolved in 0.1mol/L PBS, and finally 1% Triton X-100 is added to be mixed evenly;

s32, sample pad preparation: the sample pad treatment solution was uniformly applied to 30cm per 1mL of the pad²Drying the glass cellulose membrane at the temperature of 37 ℃ and the humidity of 10-30 percent, and sealing the bag for later use;

s4, cutting the colloidal gold combination pad, the absorbent paper and the sample pad:

cutting the colloidal gold combined pad into strips with the width of 0.3-0.5 cm by using a precise numerical control cutting machine, cutting the absorbent paper into strips with the width of 2.3-3.3 cm, and cutting the sample pad into strips with the width of 2.3-3.3 cm;

s5, assembling and cutting strips:

each component of the chromatography reagent strip is a sample pad, a colloidal gold combined pad, an NC membrane and absorbent paper, and all the components are assembled together and adhered on a polystyrene bottom plate.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts phage display technology to select a combination of binding proteins with high panning affinity and good specificity from a phage display protein library by taking plasmodium falciparum histidine-rich protein-2 (HRP2) and plasmodium vivax Lactate Dehydrogenase (LDH) as target antigens. Recombinant expression and purification are carried out by utilizing a genetic engineering technology, and the combined protein reagent replaces an antibody reagent used in malaria immunoassay to establish a colloidal gold chromatography rapid detection method. Tests show that the kit prepared by the combination of the binding protein has the storage stability far higher than that of the existing clinical detection products, has low requirements on storage conditions and low production cost, has the advantages of reliability, accuracy, safety, simplicity, convenience and stability, and has higher clinical application value.

Drawings

FIG. 1 is a schematic view of the structure of the test strip.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

Phage display library panning procedure for plasmodium falciparum L lactate dehydrogenase and histidine-rich protein 2 protein reagents:

(1) the target protein was first labeled with biotin using NHS-EZ coupling reagent (from Pierce). The biotin labeling effect was confirmed by detecting the presence of biotin by ELISA. The biotin-labeled protein was determined by adsorbing the target protein to a 96-well plate (purchased from Nunc) and then performing biotin detection using streptavidin-conjugated horseradish peroxidase (HRP). The panning process for the phage display library was as follows: a5. mu.L aliquot of the phage display library containing 10 complexity¹²Phage plasmids were mixed well with 95. mu.L of phosphate buffer containing 0.1% Tween 20 and then pre-panned 3 times for a total of 1 hour in high binding streptavidin coated plates (from Pierce). To streptavidin-coated panning wells 100. mu.L of 100nM biotinylated protein was added, the shaker speed was pre-adjusted to third (using a Heidolph VIBRAMAX 100 shaker) for 1 hour of shaking, and then the pre-panned phage was added for 2.5 hours of reaction.

(2) The elutriated wells were washed 10 times with 300. mu.L/well PBST using a plate washer (Tecan Hydroflex), followed by elution with 100. mu.L of 50mM glycine-hydrochloric acid (pH2.2) for 10 minutes, neutralization with 1M Tris-HCl (pH9.1), further elution with 100. mu.L of 100mM triethylamine for 6 minutes, and neutralization with 50. mu.L of 1M Tris-HCl (pH 7.0). The eluted phages were incubated with the ER2738 E.coli in the exponential growth phase at 37 ℃ for 1 hour at 90 rpm. The cells were plated on LB agar plates, added with 100. mu.g/mL carbenicillin, and cultured overnight at room temperature. The next day, the colonies were scraped into 5mL of 2TY medium, inoculated into 25mL of 2TY medium, added to carbenicillin (100. mu.g/mL) to an absorbance of 0.2 at a wavelength of 600nm, incubated at 37 ℃ for 1 hour at 225 rpm, followed by incubation with 10 hours⁹Complexity of M13K07 helper phage infection. After culturing at 90 rpm for 1 hour, 25. mu.g/mL kanamycin was added, and the cells were incubated at 170 rpm at 25 ℃ overnight for phagocytosisPrecipitated with 6% PEG8000, 0.3M NaCl and resuspended in 1mL of a pH8.0 buffer containing 10mM Tris, 1mM EDTA (TE buffer).

(3) mu.L of this phage suspension was taken for a second round of panning. For this round of panning, the phage display library was treated with streptavidin-labeled magnetic beads (ex Invirogen). Phage were pre-panned with 10. mu.L of washed magnetic beads on a Stuart SB2 impeller for 1 hour at 20 rpm, while the 10. mu.L of beads were labeled with 100. mu.L of 100nM biotinylated protein on the same impeller for 4 hours. Antigen-labeled magnetic beads were washed three times with PBST and then pre-panning phage was added for 1 hour. mu.L of the diluted phage and magnetic beads of PBST were added to the KingFisher 96-Flex magnetic bead extraction purification system (purchased from ThermoFisher), incubated with shaking for 1 hour, and washed 5 times with 1mL of PBST over 10 minutes. The bound phage were eluted and amplified as above. The final round of panning was treated with neutravidin high binding force plates (available from Pierce corporation) and the operation was as previously described for the first round of panning.

Example 2

The existence of phage of the target protein reagent is detected by ELISA method, and the verification result is further confirmed by sequencing, the process is as follows:

phage were recovered from wells containing the target protein, while quality control wells, which demonstrated efficient amplification of reagents directed to binding the target protein, were used to determine the amplification in the target wells. To test proteins purchased from the display library, bound target phage were detected by ELISA. Individual ER2738 colonies were purchased from infected cells from the last panning round and were selected after 6 h incubation in 100. mu.L of 2TY medium containing 100. mu.g/mL carbenicillin in 96-well plates at 37 deg.C (900 rpm). mu.L aliquots of the culture were added to 200. mu.L of 2TY medium containing carbenicillin and incubated at 37 ℃ for 1 hour (900 rpm). Then, helper phage (10. mu.L of 1011/mL) was added, followed by additional kanamycin to 25. mu.g/mL concentration, and incubated overnight at 25 deg.C (450 rpm). Streptavidin-coated plates (from Pierce) were blocked overnight at 37 ℃ with 2 × casein blocking solution (from Sigma). The next day, the closed plate was labeled with 0.4nM biotinylated target protein for 1 hour, the bacteria were centrifuged at 3000 rpm for 5 minutes, and 45. mu.L of phage-containing medium was added to wells containing biotinylated ligation vector or biotin-labeled protein for incubation for 1 hour. Reaction wells were washed 3 times with Tecan Hydroflex plate washer 300 μ L PBST, followed by addition of a wash solution containing PBST 1: 100 μ L of 1000-diluted HRP-labeled anti-phage antibody (purchased from Seramun) was reacted for 1 hour. The reaction wells were washed 10 times with 300. mu.L of PBST, and then the conjugates were detected with 100. mu.L of TMB substrate developing solution, and finally the absorbance value was measured at a wavelength of 560 nm. The color change in the reaction well containing the target protein and the negative result in the quality control well indicate that the protein binds to the antigen.

Example 3

Introducing a DNA sequence for expressing the specificity of the target protein into a target plasmid, transferring the target plasmid into escherichia coli for amplification culture, and finally testing the protein expression condition by an ELISA method after extraction and purification, wherein the method comprises the following steps:

all specific reagents were subsequently introduced into the protein expression vector for cloning and the product was determined as capture reagent in ELISA. The DNA coding sequence (SEQ ID NO: 1-4) of the binding protein reacting with the target protein is amplified through PCR, the product is treated by Nhel and Pstl restriction endonuclease, and the digested fragment is introduced into pET11a plasmid containing an expression binding protein structure and a restriction site for digestion. The clones were picked up in 5ml LB medium at 37 ℃ overnight at 225 rpm, plasmid DNA extracted with miniprep (from Qiagen) plasmid extraction kit, and then sequenced to further confirm the correct insertion site. The plasmid was transferred by heat shock into E.coli BL21(DE3) (F-ompT gal dcm lon hsdSB (rB-mB-) lambda (DE3[ lacI lacUV5-T7gene 1ind1sam7nin5]) cells and cultured overnight at 37 ℃ the next day, the culture broth was added to 10ml of LB medium, cultured at 250 rpm and 37 ℃ to an absorbance value of 0.6 at a wavelength of 600nm, then isopropyl beta-D-1 thiogalactoside (IPTG) was added to a final concentration of 1mM for 6 hours, centrifuged at 3000g after 6 hours, then resuspended in 1ml of 1 x Bugbuster (from Novagen) protein extraction reagent, nuclease was added according to the instructions given by the manufacturer, suspended for 20 minutes after mixing at room temperature, heated to 50 ℃ for 20 minutes, centrifuged at NT9400 g for 20 minutes, the supernatant was mixed with Ni-A purification resin for 50 hours, the column was washed 3 times with 30ml of a washing solution (containing 50mM50mM PBS, 500mM NaCl, 20mM imidazole, pH7.4) and then eluted with 0.1ml of an eluent (containing 50mM PBS, 500mM NaCl, 300mM imidazole, pH 7.4). The prepared binding protein is used for further pairing screening test to obtain binding proteins 1-4 for malaria detection reagent development, and the amino acid sequences of the binding proteins are shown in SEQ ID NO. 5-8.

Example 4

The preparation of the plasmodium falciparum histidine-rich protein 2(HRP2) and plasmodium vivax L Lactate Dehydrogenase (LDH) rapid detection reagent (colloidal gold method) by using the binding protein obtained by screening by using a phage display technology optimizes the process:

(1) investigation of early conditions

The effect of gold particle labeled protein is mainly in large relation with the charge of the solution, and 0.1mol/L K is used₂CO₃Adding into colloidal gold solution, adding K of 0, 5, 10, 15, 20 μ L per mL of colloidal gold solution₂CO₃The solution was then added 20. mu.g of the dialyzed two nABPs, and it was found that 20. mu.L of 0.1mol/L K was added per mL of the colloidal gold solution₂CO₃The pH value of the solution for adjusting the label can be measured by a pH test paper to be 8.6 and is close to the isoelectric point pH value of the binding protein, so that the optimal labeling condition can be formed.

(2) Binding proteins for coated and colloidal gold labeled selection and testing

The assay was performed by adjusting different concentrations of partner binding protein to select for saturated coating and a test gradient of marker concentration.

(3) Process for preparing rapid detection reagent (colloidal gold method) for plasmodium falciparum histidine-rich protein 2(HRP2) and plasmodium vivax L Lactate Dehydrogenase (LDH) by optimizing binding protein

Experiments were performed to glue binding proteins labeled with P.falciparum HRP2 and P.vivax L Lactate Dehydrogenase (LDH)And (3) optimizing the test of the body gold marker, and researching the detection sensitivity and specificity of the blood sample by changing the components of the confining liquid. The preparation process is that 0.1mol/L K is added into each milliliter of colloidal gold₂CO₃The pH was adjusted to 12. mu.L. Then adding the labeled protein to react for 40min, and adding different formula reagents to seal. After 15min of reaction, centrifugation was carried out at 10000rpm for 10 min. The supernatant was removed and 1000. mu.L of a reconstituted solution (reconstituted solution formulation: 1% BSA, 2% sucrose, dissolved in 10mmol/L phosphate buffer) was added. Then uniformly coating on 15cm²The test sample was stored in a sealed state after drying at 37 ℃ for 22 hours. The test selects the gold-labeled closed process formula containing 1% of PEG 20000. Then, comparative tests of different redissolving formulations were carried out, and the formulation containing 0.02mol/L Tris + 1% BSA was determined to be the best redissolving formulation. Finally, comparative testing studies of the treatment fluid formulations for the various sample pads were conducted, and the formulation of 0.5% BSA containing 0.1M PBS was selected as the optimal sample pad formulation.

Example 5

The plasmodium falciparum histidine-rich protein 2(HRP2) and plasmodium vivax L Lactate Dehydrogenase (LDH) rapid detection reagent (colloidal gold method) is prepared by the process of example 4 by using the binding proteins 1-4, and the process is as follows:

(1) preparation of colloidal gold bonding pad

Adding 12 mu L of 0.1mol/L potassium carbonate solution into every 1mL of colloidal gold, shaking and mixing uniformly, adding binding protein 1 and binding protein 2 for detecting plasmodium falciparum HRP2 according to 16 mu g/mL, adding binding protein 3 and binding protein 4 for detecting plasmodium vivax LDH according to 12 mu g/mL, mixing uniformly, reacting for 40min, adding 1% PEG 2000020 mu L/mL colloidal gold, mixing uniformly, sealing for 15min, centrifuging at 10000rpm for 15min, discarding supernatant, dissolving precipitate with 1000 mu L of gold-labeled solution (formula: 0.02mol/L Tris + 1% BSA + 0.1% Tween-20+ 20% sucrose), and standing at 4 ℃ for later use.

At the same time, a mouse IgG antibody-labeled colloidal gold conjugate was prepared. Adding 12 mu L of 0.1mol/L potassium carbonate solution into every 1mL of colloidal gold, shaking and mixing uniformly, adding a mouse IgG antibody according to the ratio of 20 mu g/mL, mixing uniformly, reacting for 40min, adding 1% BSA 25 mu L/mL colloidal gold, mixing uniformly, sealing for 15min, centrifuging at 10000rpm for 15min, discarding supernatant, dissolving precipitate with 1000 mu L of gold-labeled complex solution (formula: 0.02mol/L Tris + 1% BSA + 0.1% Tween-20+ 20% sucrose), and standing at 4 ℃ for later use.

Mixing the above colloidal gold conjugates, and uniformly coating on 15cm per 1mL²Dried at 37 ℃ for 22 hours and sealed for later use.

(2) Coated film scribing preparation

Preparing a coating buffer solution: 5% sucrose was added to 10mmol/L PBS (pH 7.4).

Detection line marking of plasmodium falciparum HRP 2: the conjugated protein 1 and the conjugated protein 2 are diluted to be 1mg/mL, are coated on a nitrocellulose membrane by scribing with a film spraying machine according to the scribing concentration of 0.1 muL/mm, are dried at 37 ℃ overnight, and are sealed and stored for standby.

Scribing plasmodium vivax LDH detection lines: the conjugated protein 3 and the conjugated protein 4 are diluted to be 1mg/mL, are coated on a nitrocellulose membrane by scribing with a film spraying machine according to the scribing concentration of 0.1 muL/mm, are dried at 37 ℃ overnight, and are sealed and stored for standby.

C, scribing a quality control line: the goat anti-mouse antibody (purchased from Hangzhou xian Zhi biology company) is streaked, the streaked concentration is 1.5mg/mL, the streaked concentration is 0.1 muL/mm, the streaked concentration is coated on a nitrocellulose membrane by using a film spraying machine, and the nitrocellulose membrane is dried at 37 ℃ overnight and then is sealed and stored for standby.

The streaking of the three reagents is that on the same nitrocellulose membrane, the C line, the T2 line (Plasmodium vivax LDH detection line) and the T1 line (Plasmodium falciparum HRP2 detection line) are respectively arranged from the end of water-absorbing paper in sequence.

(3) Sample pad preparation

Preparing a sample pad treatment solution: calculation 1% PVP and 0.5% BSA were weighed and dissolved in 0.1mol/L PBS, and finally 1% Triton X-100 was added and mixed.

Sample pad preparation: uniformly coating the sample pad treatment solution per 1mL on a 30cm2 glass cellulose membrane, drying in a drying oven (the temperature of the drying oven is 37 ℃, the humidity is 10-30 percent), and sealing for later use.

(4) Cutting of colloidal gold bonding pad, absorbent paper and sample pad

Cutting the colloidal gold combined pad into strips with the width of 0.4 +/-0.1 cm, cutting the absorbent paper into strips with the width of 2.8 +/-0.5 cm and cutting the sample pad into strips with the width of 2.8 +/-0.5 cm by using a precise numerical control cutting machine.

(5) Assembled slitting

After the selection and treatment of the components (sample pad, colloidal gold conjugate pad, NC membrane, absorbent paper) of the chromatographic reagent strip, the next step is to assemble the components together. The most common platform at present is a polystyrene base plate (PVC plate), which is coated with a medium-strength adhesive and covered with a protective film, similar to a double-sided tape, and the components can be bonded to the polystyrene base plate after the protective film is peeled off.

The specific process of reagent strip assembly is as follows:

firstly, a PVC plate is flatly paved on a working table.

And the protective film at the NC film pasting position on the PVC plate is slightly uncovered, and the NC film is uniformly pushed from left to right so as to be firmly pasted on the PVC plate.

And thirdly, slightly uncovering the protective film at the pasting position of the absorption pad on the PVC plate, adhering the absorption pad on the protective film, and uniformly and slightly advancing in a rolling manner to strengthen the adhesive force and prevent bubbles from being generated, wherein the absorption pad covers the NC film by 1 mm.

And fourthly, slightly uncovering the protective film at the pasting position of the colloidal gold pad at the lower edge of the NC film, and pasting the colloidal gold combined pad on the protective film, wherein the method is the same as that of the absorption pad. The colloidal gold conjugate pad was covered on the NC membrane by 1 mm.

Lightly uncovering the protective film at the lowest end of the thin plate, and adhering the sample pad on the colloidal gold bonding pad by the same method as the absorption pad. The sample pad was covered with a colloidal gold conjugate pad for 2 mm.

Cutting the adhered polystyrene sheet into reagent strips with the width of 4mm on an automatic strip cutting machine.

The resulting product is shown in fig. 1.

Example 6

Plasmodium falciparum histidine-rich protein 2(HRP 2)/plasmodium L Lactate Dehydrogenase (LDH) rapid detection reagent (colloidal gold method) performance evaluation test:

the clinical test samples studied were samples collected from clinical laboratories, and the experimental control used for the samples was gold standard microscopy for malaria detection. Compared with the control, the detection results of 156 samples are consistent with those of the control reagent, and the detection results of the other 5 samples are not consistent. Through statistical analysis, the positive coincidence rate reaches 97.9%, the negative coincidence rate reaches 95.5%, the total coincidence rate is 96.9%, the john index reaches 0.934, and the Kappa is 0.936.

The stability of the produced detection reagents was investigated and the results were as follows:

by accelerating the heat stability (45 ℃) test to the tenth week, the reagent prepared showed a slight decrease in the value of the positive blood sample. Therefore, a comparatively safe thermal stability time of eight weeks was selected, i.e., the detection reagent produced (colloidal gold method) was stable in the accelerated test at 45 ℃. It is stable for about 40 months within 8 weeks, i.e. the corresponding real-time storage temperature (22 ℃), as calculated by the arrhenius formula, whereas other immunoassays which have been used clinically have a product life mostly within 24 months. Therefore, the detection reagent prepared by the invention has longer effective period than other detection products under normal temperature storage.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> river-south university

GUANGDONG HEXIN BIOTECHNOLOGY Co.,Ltd.

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cgactaacct aggtcctatc ttaact 626

<210> 3

<211> 675

<212> DNA

<213> Plasmodium vivax

<400> 3

cattccttac tcttacttac ctcttactta cttactctta tcttcttact ttttgaaatg 60

acattgttgg actaatttcc cttgcacttt ccttgcacaa ttgacatgca ctttcactaa 120

ttactataac ttcacacact ttaacatcaa aatatgaatt ttgggtcacc aactatgtgg 180

aaaaatggtg tattgttacc caatcccaca tttatcgtac caaaaatccc ccatccacaa 240

ttttcatagg ggggggtatt gctctttggg cctaaatttg gaccattttt tcaactacca 300

cgactacttc aatcaaaagc taatactgct gcatttaggc ttcatattcc tattattatg 360

atttttggtc accctttggc cttcattttc gacgaggact ttactatttg acacaaaatt 420

gtatagacaa cattcgaaca tgtcattaca aaatttgggc tcatttatcg tacattactc 480

tcacccacaa ctaattcttt caaaatggta cttcttttaa agggggaaat tgaactggaa 540

attcgggtta tagccatttt ctacaacgtc atcaaaattt ggtacacact tcttactatt 600

catcatatag tgcatgactt atatacatac aaaatggtag catactttcc actacgttta 660

tattttgtgt gcact 675

<210> 4

<211> 748

<212> DNA

<213> Plasmodium vivax

<400> 4

tactacgtta cttctaccta ctcatttact gatacattac ttcgacttaa catcattact 60

acttctacaa ctacaacatc aatactactt ctacaatgac attaccaaca gcattactac 120

tatcttacta cgactactac tatcttacta ctactaactt acttctacat tactatgaca 180

tcagtactat ttctacaatg acattagtac tacttcaact acgacatcag tattaattct 240

aggtgctaac attagtaatg taggtcttac tttcactaac ttaggtctta actaaggtgc 300

taacattagt aatgtaggtc ttactttcac taacttaggt cttaactaat ctaacttagg 360

tcttaagttg actaacatag gtcttacttt cactaacgac cttactttca ctaatatagg 420

tcttaccttt actaacatag gtcttactac aggtcttacg ttgactaact taggtcttaa 480

cttaactaac tattaataaa ctaacatagg tcctaacatt agtaatgtag gtcttacttt 540

taatttcact aatataggtc ttaacttcac taacataggt cttaacttaa gtcttaacat 600

cactaacata ggtcctaaca ttagtaatgt aggtcttaac ttaaactaac ataggtctta 660

cttctactaa cttaggtctt aacttaacta agtaacttaa cttaacaaat ataggtccta 720

acattactaa ctttcgtctt aaggttac 748

<210> 5

<211> 194

<212> PRT

<213> Plasmodium falciparum (Plasmodium)

<400> 5

Ile Pro Tyr Ser Tyr Leu Phe Met Val Ile Cys Phe Gly Ser Tyr Gly

1 5 10 15

Leu His Leu Val Leu Leu Val Pro Tyr Tyr Tyr Ser Phe Cys His Phe

20 25 30

Arg Thr Thr His Asn His Pro Leu Phe Ile His Cys Tyr Asp Ile Val

35 40 45

Ala Leu Cys Tyr Ser Tyr Leu Val His Leu Phe Leu Thr Pro Asn Phe

50 55 60

Ile Ile Tyr Ile Ile Ile Val Val Leu Leu Gly Ser Thr Ile Ile Phe

65 70 75 80

Ile Leu Leu Gln His Ile Thr Val Phe Tyr His Leu Val Phe His Leu

85 90 95

Thr Ser Asp Val Ala Leu Tyr Leu Tyr Ala Phe Val Cys Ile Met Cys

100 105 110

Ser Phe Ile Cys Ser Cys Leu Leu Cys Thr His Phe Tyr Asn Asn Leu

115 120 125

Ile Phe Cys Ser Ala Leu Gln Gly Ile Ser Leu Leu Leu Cys Thr His

130 135 140

Ile Ser Leu Leu Phe Ala His Lys Ile Ile Thr Thr Ser Glu His Val

145 150 155 160

Ile Thr Leu Asp Thr Thr Tyr Lys Leu Ala His Phe Thr Arg Asn Asn

165 170 175

Ala Ala Gly Ala Arg Asn Leu His Val Trp Lys Phe Met Val Val Leu

180 185 190

Leu Gly

<210> 6

<211> 199

<212> PRT

<213> Plasmodium falciparum (Plasmodium)

<400> 6

Met Phe Leu Gln Thr Met Phe Asp Ile Tyr Tyr Ile Tyr Phe Ile Asp

1 5 10 15

Leu Val Leu Leu His Tyr Tyr Leu Thr Thr Thr Thr Thr Thr Leu Leu

20 25 30

Ile Asn Phe Ala Cys Ser Val Phe Asn Arg Leu Phe Tyr His Ala Thr

35 40 45

Phe Ser Tyr Gln Asn Tyr Phe Phe Asn Asp Ile Ile Thr Thr Ser Ser

50 55 60

Thr Thr Ser Leu Leu Phe Ile Thr Ile Asn Leu Val Leu His Tyr Tyr

65 70 75 80

Tyr Phe Tyr Asn Asp Ile Ile Thr Thr Cys Leu Leu Leu His Tyr Tyr

85 90 95

Asp Ile Ser Thr Ile Ser Thr Met Thr Leu Val Leu Leu Gln Leu Leu

100 105 110

Thr Thr Asn Leu Leu Ile His Tyr Phe Tyr Phe Ile Thr Asn Leu Leu

115 120 125

Leu Thr Leu Leu Leu His Tyr Tyr Tyr Asp Ile Asn Thr Thr Ser Thr

130 135 140

Thr Thr Leu Leu Leu His His His Gln His His His Asp Tyr Arg His

145 150 155 160

Asn Leu Asp Val Asp Thr Gln Val Leu Ile Ser Thr Asn Leu Gly Leu

165 170 175

Asn Leu Ala Asn Ile Gly Leu Leu Val Leu Arg Leu Leu Thr Val Ile

180 185 190

Leu Arg Leu Thr Val Leu Ser

195

<210> 7

<211> 218

<212> PRT

<213> Plasmodium vivax

<400> 7

His Ser Leu Leu Leu Leu Thr Ser Tyr Leu Leu Thr Leu Ile Phe Leu

1 5 10 15

Leu Phe Glu Met Thr Leu Leu Asp Phe Pro Leu His Phe Pro Cys Thr

20 25 30

Ile Asp Met His Phe His Leu Leu Leu His Thr Leu His Gln Asn Met

35 40 45

Asn Phe Gly Ser Pro Thr Met Trp Lys Asn Gly Val Leu Leu Pro Asn

50 55 60

Pro Thr Phe Ile Val Pro Lys Ile Pro His Pro Gln Phe Ser Gly Gly

65 70 75 80

Val Leu Leu Phe Gly Pro Lys Phe Gly Pro Phe Phe Gln Leu Pro Arg

85 90 95

Leu Leu Gln Ser Lys Ala Asn Thr Ala Ala Phe Arg Leu His Ile Pro

100 105 110

Ile Ile Met Ile Phe Gly His Pro Leu Ala Phe Ile Phe Asp Glu Asp

115 120 125

Phe Thr Ile His Lys Ile Val Thr Thr Phe Glu His Val Ile Thr Lys

130 135 140

Phe Gly Leu Ile Tyr Arg Thr Leu Leu Ser Pro Thr Thr Asn Ser Phe

145 150 155 160

Lys Met Val Leu Leu Leu Lys Gly Glu Ile Glu Leu Glu Ile Arg Val

165 170 175

Ile Ala Ile Phe Tyr Asn Val Ile Lys Ile Trp Tyr Thr Leu Leu Thr

180 185 190

Ile His His Ile Val His Asp Leu Tyr Thr Tyr Lys Met Val Ala Tyr

195 200 205

Phe Pro Leu Arg Leu Tyr Phe Val Cys Thr

210 215

<210> 8

<211> 207

<212> PRT

<213> Plasmodium vivax

<400> 8

Tyr Tyr Val Thr Ser Thr Tyr Ser Phe Thr Asp Thr Leu Leu Arg Leu

1 5 10 15

Asn Ile Ile Thr Thr Ser Thr Thr Thr Thr Ser Ile Leu Leu Leu Gln

20 25 30

His Tyr Gln Gln His Tyr Tyr Tyr Leu Thr Thr Thr Thr Thr Ile Leu

35 40 45

Leu Leu Leu Thr Tyr Phe Tyr Ile Thr Met Thr Ser Val Leu Phe Leu

50 55 60

Gln His Tyr Tyr Phe Asn Tyr Asp Ile Ser Ile Asn Ser Arg Cys His

65 70 75 80

Cys Arg Ser Tyr Phe His Leu Arg Ser Leu Arg Cys His Cys Arg Ser

85 90 95

Tyr Phe His Leu Arg Ser Leu Ile Leu Arg Ser Val Asp His Arg Ser

100 105 110

Tyr Phe His Arg Pro Tyr Phe His Tyr Arg Ser Tyr Leu Tyr His Arg

115 120 125

Ser Tyr Tyr Arg Ser Tyr Val Asp Leu Arg Ser Leu Asn Leu Leu Ile

130 135 140

Asn His Arg Ser His Cys Arg Ser Tyr Phe Phe His Tyr Arg Ser Leu

145 150 155 160

His His Arg Ser Leu Lys Ser His His His Arg Ser His Cys Arg Ser

165 170 175

Leu Lys Leu Thr Val Leu Leu Leu Leu Thr Val Leu Thr Leu Ser Asn

180 185 190

Leu Thr Gln Ile Val Leu Thr Leu Leu Thr Phe Val Leu Arg Leu

195 200 205

Claims (4)

1. The combination of the binding proteins for detecting the plasmodium falciparum HRP2 and the plasmodium vivax LDH is characterized in that the combination of the binding proteins consists of a binding protein 1 and a binding protein 2 for detecting the plasmodium falciparum HRP2, a binding protein 3 and a binding protein 4 for detecting the plasmodium vivax LDH, and the amino acid sequences of the binding proteins 1-4 are shown as SEQ ID NO. 5-8.

2. A method of preparing the binding protein combination of claim 1, comprising the steps of:

amplifying nucleotide sequences of a gene combination encoding the combination of binding proteins of claim 1, and performing double digestion on the amplified product and the pET11a plasmid by respectively adopting Nhel and Pstl restriction endonucleases; selecting clones, culturing in 5mL LB culture medium at 37 deg.C and 225 rpm overnight; the plasmid was transferred into E.coli BL21(DE3) cells by heat shock and cultured overnight at 37 ℃; the next day, the culture broth was added to 10mL of LB medium and cultured at 37 ℃ at 250 rpm to OD_600nm=0.6, isopropyl β -D-1 thiogalactoside was added to a final concentration of 1 mM; culturing the cells for 6 hours again, centrifuging at the rotating speed of 3000g, and then suspending in 1mL of 1 XBugbuster protein extraction reagent; adding nuclease, mixing at room temperature, suspending for 20 min, heating to 50 deg.C, and centrifuging at 9400g for 20 min; the supernatant and Ni-NTA purification resin 50L mixing suspension 1 hours, pH7.4 containing 50mM PBS, 500mM NaCl, 20mM imidazole 30mL washing liquid 3 times, and then pH7.4 containing 50mM PBS, 500mM NaCl, 300mM imidazole 0.1mL elution, get the claim of claim 1 binding protein combination.

3. Use of a combination of binding proteins according to claim 1 for the preparation of a reagent for the detection of malaria.

4. A method for preparing a reagent for detecting malaria, comprising the steps of:

s1, preparing a colloidal gold bonding pad:

s11, adding 12 mu L of 0.1mol/L potassium carbonate solution into every 1mL of colloidal gold, shaking and uniformly mixing, adding the binding protein 1 and the binding protein 2 for detecting the plasmodium falciparum HRP2 in the claim 1 according to 16 mu g/mL, adding the binding protein 3 and the binding protein 4 for detecting the plasmodium vivax LDH in the claim 1 according to 12 mu g/mL, uniformly mixing, reacting for 40min, adding 1% PEG 2000020 mu L/mL of colloidal gold, uniformly mixing, sealing for 15min, centrifuging at 10000rpm for 15min, discarding supernatant, dissolving precipitate with 1000 mu L of gold-labeled re-solution, and placing at 4 ℃ for later use;

s12, preparing a mouse IgG antibody labeled colloidal gold conjugate, adding 12 mu L of 0.1mol/L potassium carbonate solution into 1mL of colloidal gold, shaking and uniformly mixing, adding the mouse IgG antibody according to 20 mu g/mL, uniformly mixing, reacting for 40min, adding 1% BSA (bovine serum albumin) and 25 mu L/mL of colloidal gold, uniformly mixing, sealing for 15min, centrifuging at 10000rpm for 15min, discarding supernatant, dissolving a precipitate with 1000 mu L of gold labeled re-solution, and standing at 4 ℃ for later use; the gold-labeled complex solution consists of the following components in concentration: 0.02mol/L Tris, 1% BSA, 0.1% Tween-20 and 20% sucrose;

s13, uniformly coating the colloidal gold conjugate obtained in the steps S11 and S12 on 15cm per 1mL after mixing²Drying the glass cellulose membrane at 37 ℃ for 22 hours, and sealing for later use;

s2, coating film scribing preparation:

s21, preparing a coating buffer solution: adding 5% sucrose into 10mmol/L PBS pH 7.4;

s22, carrying out detection line marking on plasmodium falciparum HRP 2: diluting the binding protein 1 and the binding protein 2 of claim 1 to 1mg/mL, coating the diluted binding protein 1 and the binding protein 2 on a nitrocellulose membrane by using a film spraying machine according to the scribing concentration of 0.1 μ L/mm, drying the nitrocellulose membrane at 37 ℃ overnight, and sealing and storing the nitrocellulose membrane for later use;

s23, scribing plasmodium vivax LDH detection lines: diluting the binding protein 3 and the binding protein 4 of claim 1 to 1mg/mL, coating the diluted binding protein 3 and the binding protein 4 on a nitrocellulose membrane by a film spraying machine according to the scribing concentration of 0.1 μ L/mm, drying the nitrocellulose membrane at 37 ℃ overnight, and sealing and storing the nitrocellulose membrane for later use;

s24, scribing a quality control line C: scribing goat anti-mouse antibody with a scribing concentration of 1.5mg/mL according to a scribing concentration of 0.1 muL/mm, scribing and coating on a nitrocellulose membrane by using a film spraying machine, drying at 37 ℃ overnight, and sealing and storing for later use;

scribing the three reagents of the steps S22-S24 on the same nitrocellulose membrane, and sequentially starting from the end of water-absorbing paper to form a C line, a P.vivax LDH detection line T2 line and a P.falciparum HRP2 detection line T1 line;

s3, preparing a sample pad:

s31, preparing a sample pad treating fluid: 1% PVP and 0.5% BSA are calculated and weighed to be dissolved in 0.1mol/L PBS, and finally 1% Triton X-100 is added to be mixed evenly;

s32, sample pad preparation: the sample pad treatment solution was uniformly applied to 30cm per 1mL of the pad²Drying the glass cellulose membrane at the temperature of 37 ℃ and the humidity of 10-30 percent, and sealing the bag for later use;

s4, cutting the colloidal gold combination pad, the absorbent paper and the sample pad:

cutting the colloidal gold bonding pad into strips with the width of 0.3-0.5 cm by using a precise numerical control cutting machine, cutting the absorbent paper into strips with the width of 2.3-3.3 cm, and cutting the sample pad into strips with the width of 2.3-3.3 cm;

s5, assembling and cutting strips:

each component of the chromatography reagent strip is a sample pad, a colloidal gold combined pad, an NC membrane and absorbent paper, and all the components are assembled together and adhered on a polystyrene bottom plate.

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