CN112816700A

CN112816700A - Colloidal gold test strip for detecting HIV antibody in urine

Info

Publication number: CN112816700A
Application number: CN202011576176.3A
Authority: CN
Inventors: 吴滨; 曹丹琴; 王立童; 吴静; 敖翔; 胡祥叶
Original assignee: Hangzhou Xinmai Biotechnology Co Ltd
Current assignee: Hangzhou Xinmai Biotechnology Co Ltd
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2021-05-18

Abstract

The invention discloses a colloidal gold test strip for detecting HIV antibodies in urine, which comprises a plastic base plate, a sample pad, a nitrocellulose membrane, absorbent paper and a gold label pad, wherein the absorbent paper, the nitrocellulose membrane, the gold label pad and the sample pad are all fixed on the plastic base plate, a colloidal gold-labeled SPA and HIV gp41 fusion antigen is solidified on the gold label pad, two ends of the nitrocellulose membrane are respectively lapped with the inner end of the absorbent paper and the inner end of the gold label pad, the inner end of the sample pad is lapped with the outer end of the gold label pad, a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is mixed and coated with HIV gp41 recombinant antigen and gp36 recombinant antigen, and the quality control line is coated with sheep polyclonal antibody. The invention is used for primary screening of AIDS detection, has convenient and rapid operation, non-invasive sampling and high detection sensitivity, and is suitable for self-detection of common people.

Description

Colloidal gold test strip for detecting HIV antibody in urine

Technical Field

The invention relates to a test strip, in particular to a colloidal gold test strip for detecting HIV antibodies in urine.

Background

The human immunodeficiency virus is also called AIDS virus, and is a retrovirus causing defects of the human immune system. This virus attacks and gradually destroys the human immune system, rendering the host unprotected when infected. The large-scale HIV detection is a serious challenge to manpower, material resources, financial resources and time, and particularly, the development and innovation of HIV detection technology are required to be accurate, rapid and non-invasive along with the global spread of AIDS and the risk cases that medical personnel are infected.

The current AIDS diagnosis program is to carry out western blot diagnosis after the AIDS diagnosis program is primarily screened to be positive. The primary screening method comprises two main detection methods:

1. blood detection: the method mainly comprises a chemiluminescence method, a time-resolved fluorescence method and an immunochromatography (colloidal gold method or latex method), and the methods judge whether the HIV infection exists by collecting blood samples of patients and qualitatively or quantitatively detecting human immunodeficiency virus (HIV-1/HIV-2) antibodies in human serum, plasma and whole blood in vitro.

However, both chemiluminescence and time-resolved fluorescence in blood detection methods require large-scale equipment, and are only equipped in hospitals at or above the county level. And both methods are time consuming and require specialized technical operators. In addition, the blood detection is a traumatic detection, and the blood transmission is one of 3 major ways for the transmission of AIDS, and the defects of the blood detection method are that: (1) the risk of medical personnel being punctured and infected during the blood drawing process; (2) the risk of infection exists in the garbage and treatment of various related medical instruments after blood drawing; (3) the patient can not draw blood to detect by oneself, thereby ensuring personal privacy

2. And (3) detecting oral mucosa exudate: the main method is immunochromatography (colloidal gold method or latex method). Namely, the human immunodeficiency virus HIV-1/2 antibody in the human oral mucosa exudate is detected by the exudate after the oral swab is continuously wiped on the gum line. This method is much less risky than blood transmission, but still presents a high risk of transmission such as aids exposure.

The presence of HIV-specific antibodies in urine has been extensively studied and confirmed. The urine detection method without/with extremely weak infectivity only has enzyme-linked immunosorbent assay approved by CFDA at present, but the method consumes long time (3 h); the colloidal gold method which can also carry out qualitative detection is popular due to simple operation and short time consumption, but has the problem of low sensitivity due to low urine antibody content, so that the existing market has few products and most products have poor performance. In addition, urine tests have reported a higher problem of false positives in saliva compared to blood.

In addition, the traditional colloidal gold method for detecting urine HIV antibody test paper strip adopts a double-antigen sandwich method alone, and the method has poor detection sensitivity; there are also methods for detecting urine HIV antibodies by combining the double antigen sandwich method with an indirect method, but this method requires separate labeling of the two proteins, increasing the workload and cost.

Disclosure of Invention

The invention aims to solve the problems of the HIV primary screening detection method in the prior art, and provides a colloidal gold test strip for detecting HIV antibodies in urine, which is used for primary screening of AIDS detection, is convenient and rapid to operate, has non-invasive sampling and high detection sensitivity, and is suitable for self-detection of common people.

In order to achieve the purpose, the invention adopts the following technical scheme: a colloidal gold test strip for detecting HIV antibodies in urine comprises a plastic base plate, a sample pad, a nitrocellulose membrane, absorbent paper and a gold label pad, wherein the absorbent paper, the nitrocellulose membrane, the gold label pad and the sample pad are all fixed on the plastic base plate, a colloidal gold-labeled SPA and HIV gp41 fusion antigen is solidified on the gold label pad, two ends of the nitrocellulose membrane are respectively lapped with the inner end of the absorbent paper and the inner end of the gold label pad, the inner end of the sample pad is lapped with the outer end of the gold label pad, a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is mixed and coated with HIV gp41 recombinant antigen and gp36 recombinant antigen, and the quality control line is coated with sheep polyclonal antibody. Based on the technical problems of the existing HIV primary screening detection method, the invention designs a novel test strip for detecting urine HIV antibodies based on a colloidal gold method by constructing a recombinant antigen fusing the preferred epitope of SPA and the preferred epitope of HIV gp41 antigen, and the test strip is combined and detected by a double-antigen sandwich method and an indirect method, is convenient and rapid to operate, has the effects of noninvasive sampling, can reduce the labeling times and the gold spraying times of colloidal gold, reduces the workload and the cost, improves the sensitivity, can be used for the primary screening of AIDS detection, and is suitable for the self-detection of common people.

Preferably, the absorbent paper, the sample pad and the gold label pad are provided with protective films.

Preferably, the sample pad is prepared by the following method: and (3) cutting the glass fiber into required specifications, uniformly spraying the glass fiber with the treatment solution, and drying. The drying step is drying in a 37 ℃ oven for 12-16 h.

Preferably, the treatment liquid is prepared by the following method: adding 1.2g of Tris, 1g of sodium carbonate, 0.5g of Casien Na, 1g of PVP, 0.5g of HPMC, 1g of beta-cyclodextrin, 2g of surfactant S9 and 0.05g of preservative Proclin300 into 80ml of ultrapure water, stirring until the mixture is completely dissolved, adjusting the pH value to 8.5 by using 1M hydrochloric acid, and adding ultrapure water to fix the volume to 100 ml.

Preferably, the nitrocellulose membrane is prepared by the following method: flatly pasting a nitrocellulose membrane on a plastic base plate, respectively diluting the HIV gp41 recombinant antigen to the concentration of 2.0mg/ml, diluting the gp36 recombinant antigen to the concentration of 1.0mg/ml, diluting the goat polyclonal antibody to the concentration of 0.5mg/ml, uniformly coating the diluted solution mixed with the HIV gp41 recombinant antigen and the gp36 recombinant antigen on a detection line of the nitrocellulose membrane by a three-dimensional dot-film metal spraying instrument in a spraying amount of 1 mu l/cm, uniformly coating the diluted goat polyclonal antibody solution on a quality control line of the nitrocellulose membrane in a spraying amount of 1 mu l/cm, and drying. The drying step is drying in a 37 ℃ oven for 12-16 h.

Preferably, the coating buffer is prepared by the following method: 80ml of ultrapure water was added with 1.79g of Na₂HPO₄·12H₂O, 0.9g of NaCl and 0.05g of a preservative Proclin300, stirring until the components are completely dissolved, adjusting the pH to 8.0 by using 1M hydrochloric acid, and adding ultrapure water to make the volume constant to 100 ml.

Preferably, the gold-labeled pad is prepared by the following method:

(1) measuring a colloidal gold solution with the particle size of 40-50 nm by using a pipette, adding 1% of 0.1M PBS (phosphate buffer solution) with the pH value of 7.6 according to the volume of the colloidal gold solution, stirring on a magnetic stirrer, adjusting the pH value to 8.5 by using 1M NaOH, taking SPA and HIV gp41 fusion protein, marking the colloidal gold according to the ratio of 15ug/ml gold water, quickly adding the colloidal gold into the colloidal gold solution at one time, continuously and quickly stirring for 30min, adding a sealant BSA (bovine serum albumin), keeping the final concentration of the sealant at 5mg/ml, continuously stirring for 20min, centrifuging for 20min at 10 ℃ at 10000r/min, removing the supernatant, re-suspending the colloidal gold complex solution to the original volume of 10%, and uniformly mixing by vortex to obtain a colloidal gold-fusion protein conjugate;

(2) and (3) uniformly spraying the colloidal gold-fusion protein conjugate on a gold label pad by using a three-dimensional dot film gold spraying instrument with the spraying amount of 8ul/cm, and drying. The drying step is drying in a 37 ℃ oven for 2-4 h.

Preferably, the gold-labeled complex solution is prepared by the following method: 0.6g of Tris, 0.5g of Casein Na, 1g of PVP, 0.5g of PEG20000, 1g of surfactant S9, 20g of cane sugar and 0.05g of preservative Proclin300 are added into 80ml of ultrapure water, stirred until the mixture is completely dissolved, the pH value is adjusted to 8.0 by using 1M hydrochloric acid, and the volume is adjusted to 100ml by adding the ultrapure water.

Preferably, the SPA and HIV gp41 fusion protein is prepared by the following method:

(a) respectively taking SPA protein and HIV gp41 protein as target antigens, respectively analyzing the hydrophilicity and antigenicity of two antigen epitope sequences by using biological software DNAssist2.0, selecting a section of dominant sequence of SPA protein and a section of dominant sequence of HIV gp41 protein, and connecting the two by a flexible segment consisting of 4 glycines to obtain the polypeptide shown in SEQ ID No: 1, SPA and gp41 fusion protein amino acid sequence;

(b) according to the preferred codon of the escherichia coli, the amino acid sequence of the SPA and gp41 fusion protein is converted into a corresponding nucleotide sequence, and the obtained SPA and gp41 fusion protein nucleotide sequence is shown as SEQ ID No: 2, respectively adding enzyme cutting sites BamHI and EcoRI at the upper and lower reaches of the corresponding nucleotide sequence, synthesizing a target gene, and cloning the target gene into a pMD19-T vector; (c) the pMD19-T vector containing the target gene and the pET-28a (+) vector are respectively cut by restriction enzymes BamHI and EcoRI for 12h at 37 ℃, the cut products are respectively electrophoresed through 1% agarose gel, the target gene and the pET-28a (+) vector are recovered by cutting gel, the recovered target gene and the pET-28a (+) vector are connected for 12h at 4 ℃ by using T4 ligase according to the proportion of 7:1, and then the connection product is transformed into DH5 alpha competent cells, and spread on an LB plate containing 50. mu.g/mL kanamycin resistance, incubated at 37 ℃ for 12 hours, selecting the monoclonal strain to LB liquid culture medium containing 50 mug/mL kanamycin resistance, culturing for 12h at 37 ℃ by a constant temperature shaking table, extracting plasmids by adopting a plasmid purification kit, and obtaining a correct recombinant expression vector after BamHI and EcoRI double enzyme digestion identification;

(d) transforming E.coli ER2566 competent cells by the constructed recombinant expression vector, coating the competent cells on an LB plate containing 50 mu g/mL kanamycin resistance, and culturing the competent cells at 37 ℃ overnight; the second day, selecting a monoclonal strain on the plate to an LB liquid culture medium containing 50 microgram/mL kanamycin resistance, carrying out shake culture at a constant temperature of 37 ℃ for 8 hours, adding an inducer isopropyl sulfo-beta-D-galactoside to a final concentration of 1.0mmol/L, preparing a protein electrophoresis sample after carrying out induced expression for 4 hours, wherein a 10% polyacrylamide gel electrophoresis result shows that the SPA and HIV gp41 fusion protein is successfully expressed, and obtaining a fusion protein expression strain;

(c) inoculating a fusion protein expression strain to an LB liquid culture medium, adding kanamycin to the final concentration of 50 mu g/mL, culturing for 8 hours at 37 ℃ by using a constant temperature shaking table, and then, adding the strain into the LB liquid culture medium containing 50 mu g/mL of kanamycin according to the weight ratio of 1: diluting in a proportion of 100, subpackaging into a bacteria culture bottle, placing into a constant temperature shaker at 37 ℃ for culturing until OD600 is 0.8, adding an inducer isopropyl sulfo-beta-D-galactoside until the final concentration is 1.0mmol/L, continuing culturing and inducing for 4h, centrifuging to collect thalli, performing low-temperature ultrasonic bacteria breaking, centrifuging at low temperature, taking the supernatant, passing through a nickel agarose affinity chromatography column, washing and eluting to finally obtain the SPA and HIV gp41 fusion protein.

Therefore, the invention has the following beneficial effects:

(1) the test strip is used for detecting urine samples and sampling without wound, and cross infection caused by using modes such as blood sampling and the like is avoided. The test strip can be directly inserted into a sample to be tested, so that a user does not need to contact the sample in the whole process, the operation is simple, and the detection is convenient;

(2) the test strip disclosed by the invention is combined with an indirect method and a double-antigen sandwich method, so that the detection sensitivity is improved, the test strip is suitable for personal auxiliary detection, and the test strip plays a role in self-detection screening during medical observation and medical detection;

(3) by preparing the SPA and HIV gp41 fusion antigen for colloidal gold labeling, compared with the traditional SPA, HIV antigen is labeled separately, the labeling times and gold spraying times are reduced, the material loss and manpower are reduced, the cost is reduced, and the effect of improving the sensitivity by combining an indirect method and a double-antigen sandwich method for detection is achieved.

Drawings

FIG. 1 is a schematic diagram of an embodiment of the present invention.

In the figure: the device comprises a plastic bottom plate 1, a sample pad 2, a nitrocellulose membrane 3, absorbent paper 4, a gold label pad 5, a detection line 6, a quality control line 7 and a protection film 8.

Detailed Description

The invention is further described with reference to the following figures and detailed description.

The colloidal gold test strip for detecting the HIV antibody in urine as shown in figure 1 comprises a plastic base plate 1, a sample pad 2, a nitrocellulose membrane 3, absorbent paper 4 and a gold label pad 5, wherein the absorbent paper, the nitrocellulose membrane, the gold label pad and the sample pad are all fixed on the plastic base plate, a colloidal gold-labeled SPA and HIV gp41 fusion antigen is solidified on the gold label pad, two ends of the nitrocellulose membrane are respectively lapped with the inner end of the absorbent paper and the inner end of the gold label pad, the inner end of the sample pad is lapped with the outer end of the gold label pad, a detection line 6 and a quality control line 7 are arranged on the nitrocellulose membrane, the detection line is mixed and coated with an HIV gp41 recombinant antigen and a gp36 recombinant antigen, and the quality control line is coated with sheep polyclonal antibody.

The preparation method of the colloidal gold test strip for detecting the HIV antibody in urine comprises the following steps: taking a plastic bottom plate stuck with a nitrocellulose membrane, sticking a gold label pad on the plastic bottom plate close to one side of a detection line, overlapping the inner end of the gold label pad with the nitrocellulose membrane by about 0.5mm, sticking a sample pad on the gold label pad and 0.5mm with the outer end of the gold label pad, sticking absorbent paper 6 on the plastic bottom plate close to one side of a quality control line and overlapping the absorbent paper with the nitrocellulose membrane by about 0.5mm, covering a protective film on the gold label pad and the sample pad and overlapping the nitrocellulose membrane 5 by about 1mm, covering the protective film on the absorbent paper and overlapping the absorbent paper by about 1mm, and placing the assembled plastic bottom plate 2 on a high-precision automatic slitter to be cut into 3mm test strips;

the sample pad in the colloidal gold test strip for detecting the HIV antibody in urine is prepared by the following method: cutting glass fiber into a sheet with a width of 16mm and a length of 30cm, placing the sheet on a net rack, uniformly spraying 3.5ml of treatment solution on each glass fiber, placing the sheet in a 37 ℃ oven, and drying for 12 hours, wherein the sample pad treatment solution is prepared as follows: adding 1.2g of Tris, 1g of sodium carbonate, 0.5g of Casien Na, 1g of PVP, 0.5g of HPMC, 1g of beta-cyclodextrin, 2g S9 and 0.05g of preservative Proclin300 into 80ml of ultrapure water, stirring until the materials are completely dissolved, adjusting the pH value to 8.5 by using 1M hydrochloric acid, and adding ultrapure water to fix the volume to 100ml to obtain a sample pad treatment solution;

the nitrocellulose membrane in the colloidal gold test strip for detecting the HIV antibody in urine is prepared by the following method: flatly sticking a nitrocellulose membrane on a plastic bottom plate, respectively diluting HIV gp41 recombinant antigen to the concentration of 2.0mg/ml, diluting gp36 recombinant antigen to the concentration of 1.0mg/ml and sheep polyclonal antibody to the concentration of 0.5mg/ml by using a coating buffer solution, uniformly coating a diluted solution mixed with HIV gp41 recombinant antigen and gp36 recombinant antigen on a detection line of a nitrocellulose membrane by using a three-dimensional dot-film metal spraying instrument in a spraying amount of 1 mu l/cm, uniformly coating a diluted sheep polyclonal antibody solution on a quality control line of the cellulose membrane in a spraying amount of 1 mu l/cm, controlling the distance between the quality control line and the detection line to be 0.5cm, and drying in a drying oven at 37 ℃ for 12 hours, wherein the coating buffer solution is prepared by the following method: 80ml of ultrapure water was added with 1.79g of Na₂HPO₄·12H₂O, 0.9g of NaCl and 0.05g of a preservative Proclin300, stirring until the NaCl and the Proclin are completely dissolved, adjusting the pH value to 8.0 by using 1M hydrochloric acid, and adding ultrapure water to a constant volume of 100 ml;

the gold-labeled pad in the colloidal gold test strip for detecting the HIV antibody in urine is prepared by the following method:

(1) measuring a colloidal gold solution with the particle size of 40-50 nm by using a pipette, adding 1% of 0.1M PBS (phosphate buffer solution) with the pH value of 7.6 according to the volume of the colloidal gold solution, stirring on a magnetic stirrer, adjusting the pH value to 8.5 by using 1M NaOH, taking SPA and HIV gp41 fusion protein, marking the colloidal gold according to the ratio of 15ug/ml gold water, quickly adding the colloidal gold into the colloidal gold solution at one time, continuously and quickly stirring for 30min, adding a sealant BSA (bovine serum albumin), keeping the final concentration of the sealant at 5mg/ml, continuously stirring for 20min, centrifuging for 20min at 10 ℃ at 10000r/min, removing the supernatant, re-suspending the colloidal gold complex solution to the original volume of 10%, and uniformly mixing by vortex to obtain a colloidal gold-fusion protein conjugate; the gold-labeled complex solution is prepared by the following method: adding 0.6g of Tris, 0.5g of Casein Na, 1g of PVP, 0.5g of PEG20000, 1g of surfactant S9, 20g of cane sugar and 0.05g of preservative Proclin300 into 80ml of ultrapure water, stirring until the mixture is completely dissolved, adjusting the pH to 8.0 by using 1M hydrochloric acid, and adding ultrapure water to fix the volume to 100 ml; the SPA and HIV gp41 fusion protein is prepared by the following method:

(b) the nucleotide sequence of the SPA and gp41 fusion protein obtained by converting the amino acid sequence of the fusion protein into the corresponding nucleotide sequence according to the preferred codon of escherichia coli is shown as SEQ ID No: 2, respectively adding enzyme cutting sites BamHI and EcoRI at the upper and lower reaches of the corresponding nucleotide sequence, synthesizing a target gene, and cloning the target gene into a pMD19-T vector;

(c) the pMD19-T vector containing the target gene and the pET-28a (+) vector are respectively cut by restriction enzymes BamHI and EcoRI for 12h at 37 ℃, the cut products are respectively electrophoresed through 1% agarose gel, the target gene and the pET-28a (+) vector are recovered by cutting gel, the recovered target gene and the pET-28a (+) vector are connected for 12h at 4 ℃ by using T4 ligase according to the proportion of 7:1, and then the connection product is transformed into DH5 alpha competent cells, and spread on an LB plate containing 50. mu.g/mL kanamycin resistance, incubated at 37 ℃ for 12 hours, selecting the monoclonal strain to LB liquid culture medium containing 50 mug/mL kanamycin resistance, culturing for 12h at 37 ℃ by a constant temperature shaking table, extracting plasmids by adopting a plasmid purification kit, and obtaining a correct recombinant expression vector after BamHI and EcoRI double enzyme digestion identification;

(2) The colloidal gold-fusion protein conjugate is uniformly sprayed on a gold label pad with the width of 6mm and the length of 30cm by a three-dimensional dot-film gold spraying instrument in a spraying amount of 8ul/cm, and the gold label pad is placed in an oven to be dried for 4 hours at 37 ℃.

The use method of the colloidal gold test strip for detecting the HIV antibody in urine comprises the following steps: placing a sample to be detected, a detection test strip and other detection materials at room temperature for balancing; and inserting the sample immersion end of the test strip into a urine specimen container, taking out and horizontally placing the test strip when the liquid moves to the membrane, and reading for 15 min. If the sample contains HIV antibody, the sample is firstly combined with the fusion antigen of SPA and gp41 labeled by colloidal gold specifically, and then is chromatographed upwards under the capillary force to be combined with HIV gp41 recombinant antigen coated on the nitrocellulose membrane to form a solid phase antigen-HIV antibody-colloidal gold marker ternary complex, so that a red strip is displayed on the detection line (T). If the sample is negative, no red band will appear. The nitrocellulose membrane is also coated with goat polyclonal antibody as a quality control line (C), and the goat polyclonal antibody can be combined with the colloidal gold marker no matter whether HIV antibody exists in the sample or not, and a red strip appears on the quality control line as the basis of experimental effectiveness.

Sensitivity, specificity assay:

the test paper strip is used for testing HIV urine antibody reference substances (urine rapid reagents) of China pharmaceutical and biological product institute, and the results are shown in Table 1.

Table 1 national reference disc test results

Note: "-" indicates negative; "+" indicates positive.

The results in Table 1 show that the test strip of the invention has the sensitivity of detecting urine HIV antibody of more than or equal to 99 percent and the specificity of detecting urine HIV antibody of more than or equal to 99 percent, and meets the HIV antibody detection standard of China pharmaceutical and biological product testing institute.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

SEQUENCE LISTING

<110> Hangzhou Xinmai Biotechnology Co., Ltd

<120> colloidal gold test strip for detecting HIV antibody in urine

<130> 20201212

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 334

<212> PRT

<213> Artificial Synthesis

<220>

<223> SPA and gp41 fusion protein amino acid sequence

<400> 1

Phe Asn Lys Asp Gln Gln Ser Ala Phe Tyr Glu Ile Leu Asn Met Pro

1 5 10 15

Asn Leu Asn Glu Ala Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys Asp

20 25 30

Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn

35 40 45

Glu Ser Gln Ala Pro Lys Ala Asp Asn Phe Asn Lys Glu Gln Gln Asn

50 55 60

Ala Phe Tyr Glu Ile Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Gly

65 70 75 80

Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu

85 90 95

Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ala Asp Asn

100 105 110

Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu

115 120 125

Pro Asn Leu Thr Glu Glu Gln Arg Asn Gly Phe Ile Gln Ser Leu Lys

130 135 140

Asp Asp Pro Ser Val Ser Lys Glu Ile Leu Ala Glu Ala Lys Lys Leu

145 150 155 160

Asn Asp Ala Gln Ala Pro Lys Glu Glu Asp Asn Asn Lys Pro Gly Lys

165 170 175

Glu Asp Asn Asn Lys Pro Gly Lys Glu Asp Asn Asn Lys Pro Gly Lys

180 185 190

Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Asn Lys Lys Pro Gly Lys

195 200 205

Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Asn Lys Lys Pro Gly Lys

210 215 220

Glu Asp Gly Asn Lys Pro Gly Lys Glu Asp Gly Asn Gly Gly Gly Gly

225 230 235 240

Ala Val Glu Arg Tyr Leu Gln Asp Gln Gln Leu Leu Gly Ile Trp Gly

245 250 255

Cys Ser Gly Lys Leu Ile Cys Thr Thr Ser Val Pro Trp Asn Thr Ser

260 265 270

Trp Ser Asn Arg Ser Gln Asp Gln Ile Trp Lys Asn Met Thr Trp Met

275 280 285

Gln Trp Glu Arg Glu Ile Glu Asn Tyr Thr Asn Glu Ile Tyr Thr Leu

290 295 300

Ile Glu Gln Ser Gln Asn Gln Gln Glu Lys Asn Glu Leu Asp Leu Leu

305 310 315 320

Lys Leu Asp Glu Trp Ala Ser Leu Trp Asn Trp Phe Asn Ile

325 330

<210> 2

<211> 1002

<212> DNA

<213> Artificial Synthesis

<220>

<223> SPA and gp41 fusion protein nucleotide sequence

<400> 2

tttaacaaag atcagcagag cgccttttat gaaattctga acatgccgaa cctgaacgaa 60

gcccagcgca acggctttat tcagagcctg aaagatgatc cgagccagag caccaacgtg 120

ctgggcgaag ccaaaaaact gaacgaaagc caggccccga aagccgataa ctttaacaaa 180

gaacagcaga acgcctttta tgaaattctg ccgaacctga acgaagaaca gcgcaacggc 240

tttattcaga gcctgaaaga tgatccgagc cagagcgcca acctgctggc cgaagccaaa 300

aaactgaacg atgcccaggc cccgaaagcc gataacaaat ttaacaaaga acagcagaac 360

gccttttatg aaattctgca tctgccgaac ctgaccgaag aacagcgcaa cggctttatt 420

cagagcctga aagatgatcc gagcgtgagc aaagaaattc tggccgaagc caaaaaactg 480

aacgatgccc aggccccgaa agaagaagat aacaacaaac cgggcaaaga agataacaac 540

aaaccgggca aagaagataa caacaaaccg ggcaaagaag atggcaacaa accgggcaaa 600

gaagataaca aaaaaccggg caaagaagat ggcaacaaac cgggcaaaga agataacaaa 660

aaaccgggca aagaagatgg caacaaaccg ggcaaagaag atggcaacgg cggcggcggc 720

gccgtggaac gctatctgca ggatcagcag ctgctgggca tttggggctg cagcggcaaa 780

ctgatttgca ccaccagcgt gccgtggaac accagctgga gcaaccgcag ccaggatcag 840

atttggaaaa acatgacctg gatgcagtgg gaacgcgaaa ttgaaaacta taccaacgaa 900

atttataccc tgattgaaca gagccagaac cagcaggaaa aaaacgaact ggatctgctg 960

aaactggatg aatgggccag cctgtggaac tggtttaaca tt 1002

Claims

1. The colloidal gold test strip for detecting the HIV antibody in urine is characterized by comprising a plastic base plate (1), a sample pad (2), a nitrocellulose membrane (3), absorbent paper (4) and a gold label pad (5), wherein the absorbent paper, the nitrocellulose membrane, the gold label pad and the sample pad are fixed on the plastic base plate, a colloidal gold-labeled SPA and HIV gp41 fusion antigen is solidified on the gold label pad, two ends of the nitrocellulose membrane are respectively lapped with the inner end of the absorbent paper and the inner end of the gold label pad, the inner end of the sample pad is lapped with the outer end of the gold label pad, a detection line (6) and a quality control line (7) are arranged on the nitrocellulose membrane, the detection line is mixed and coated with an HIV gp41 recombinant antigen and a gp36 recombinant antigen, and the quality control line is coated with a sheep polyclonal antibody.

2. The colloidal gold test strip for detecting HIV antibody in urine according to claim 1, wherein the absorbent paper, the sample pad and the gold label pad are provided with a protective film (8).

3. The method of claim 1, wherein the sample pad is prepared by the following steps: and (3) cutting the glass fiber into required specifications, uniformly spraying the glass fiber with the treatment solution, and drying.

4. The test strip of colloidal gold for detecting HIV antibody in urine as claimed in claim 3, wherein the treating solution is prepared by the following steps: adding 1.2g of Tris, 1g of sodium carbonate, 0.5g of Casien Na, 1g of PVP, 0.5g of HPMC, 1g of beta-cyclodextrin, 2g of surfactant S9 and 0.05g of preservative Proclin300 into 80ml of ultrapure water, stirring until the components are completely dissolved, adjusting the pH value to 8.5 by using 1M hydrochloric acid, and adding ultrapure water to fix the volume to 100 ml.

5. The test strip for detecting HIV antibody in urine according to claim 1, wherein the nitrocellulose membrane is prepared by the following steps: flatly pasting a nitrocellulose membrane on a plastic base plate, respectively diluting the HIV gp41 recombinant antigen to the concentration of 2.0mg/ml, diluting the gp36 recombinant antigen to the concentration of 1.0mg/ml, diluting the goat polyclonal antibody to the concentration of 0.5mg/ml, uniformly coating the diluted solution mixed with the HIV gp41 recombinant antigen and the gp36 recombinant antigen on a detection line of the nitrocellulose membrane by a three-dimensional dot-film metal spraying instrument in a spraying amount of 1 mu l/cm, uniformly coating the diluted goat polyclonal antibody solution on a quality control line of the nitrocellulose membrane in a spraying amount of 1 mu l/cm, and drying.

6. The test strip of colloidal gold for detecting HIV antibody in urine as claimed in claim 5, wherein said coating buffer is prepared by the following steps: 80ml of ultrapure water was added with 1.79g of Na₂HPO₄·12H₂O, 0.9g of NaCl and 0.05g of a preservative Proclin300, stirring until the components are completely dissolved, adjusting the pH to 8.0 by using 1M hydrochloric acid, and adding ultrapure water to make the volume constant to 100 ml.

7. The method of claim 1, wherein the gold-labeled pad is prepared by the following steps:

(1) measuring a colloidal gold solution with the particle size of 40-50 nm of the required labeling amount by using a pipette, adding 1% of 0.1M PBS solution with the pH value of 7.6 according to the volume of the colloidal gold solution, stirring on a magnetic stirrer while adjusting the pH value to 8.5 by using 1M NaOH, taking SPA and HIV gp41 fusion protein, labeling the colloidal gold according to the ratio of 15ug/ml gold water, quickly adding the colloidal gold into the colloidal gold solution at one time, continuously and quickly stirring for 30min, adding a sealant BSA with the final concentration of 5mg/ml, continuously stirring for 20min, centrifuging at 10 ℃ for 20min at 10000r/min, removing the supernatant, re-suspending the colloidal gold solution to the original volume of 10%, and uniformly mixing by vortex to obtain a colloidal gold-fusion protein conjugate;

(2) and (3) uniformly spraying the colloidal gold-fusion protein conjugate on a gold label pad by using a three-dimensional dot film gold spraying instrument with the spraying amount of 8ul/cm, and drying.

8. The test strip of colloidal gold for detecting HIV antibody in urine as claimed in claim 7, wherein said gold-labeled solution is prepared by the following steps: 0.6g of Tris, 0.5g of Casein Na, 1g of PVP, 0.5g of PEG20000, 1g of surfactant S9, 20g of cane sugar and 0.05g of preservative Proclin300 are added into 80ml of ultrapure water, stirred until the components are completely dissolved, the pH value is adjusted to 8.0 by using 1M hydrochloric acid, and the volume is adjusted to 100ml by adding the ultrapure water.

9. The test strip of colloidal gold for detecting HIV antibody in urine as claimed in claim 7, wherein the fusion protein of SPA and HIV gp41 is prepared by the following steps:

(a) respectively taking SPA protein and HIV gp41 protein as target antigens, respectively analyzing the hydrophilicity and antigenicity of two antigen epitope sequences by using biological software DNAssist2.0, selecting a segment of dominant sequence of SPA protein and a segment of dominant sequence of HIV gp41 protein, and connecting the two by a flexible segment consisting of 4 glycines to obtain a protein sequence shown as SEQ ID No: 1, SPA and gp41 fusion protein amino acid sequence;

(b) converting the amino acid sequence of the fusion protein into a corresponding nucleotide sequence according to the preferred codon of the escherichia coli, and obtaining the nucleotide sequence of the SPA and gp41 fusion protein as shown in SEQ ID No: 2, respectively adding enzyme cutting sites BamHI and EcoRI at the upper and lower reaches of the corresponding nucleotide sequence, synthesizing a target gene, and cloning the target gene into a pMD19-T vector;

(d) e.coli ER2566 competent cells are transformed by the constructed recombinant expression vector, and are coated on an LB plate containing 50 mu g/mL kanamycin resistance, and are cultured overnight at 37 ℃; the second day, picking the monoclonal strain on the plate to LB liquid culture medium containing 50 mug/mL kanamycin resistance, after shaking culture at 37 ℃ for 8h, adding an inducer isopropyl sulfo-beta-D-galactoside to the final concentration of 1.0mmol/L, preparing a protein electrophoresis sample after induced expression for 4h, wherein the result of 10% polyacrylamide gel electrophoresis shows that the SPA and HIV gp41 fusion protein is successfully expressed, and obtaining a fusion protein expression strain;

(c) inoculating a fusion protein expression strain to an LB liquid culture medium, adding kanamycin to the final concentration of 50 mu g/mL, carrying out shake culture at the constant temperature of 37 ℃ for 8 hours, and then, adding the strain into the LB liquid culture medium containing 50 mu g/mL kanamycin to perform the following steps of: diluting in a proportion of 100, subpackaging into a bacteria culture bottle, placing into a constant temperature shaker at 37 ℃ for culturing until OD600 is 0.8, adding an inducer isopropyl sulfo-beta-D-galactoside until the final concentration is 1.0mmol/L, continuing culturing and inducing for 4h, centrifuging to collect thalli, performing low-temperature ultrasonic bacteria breaking, centrifuging at low temperature, taking the supernatant, passing through a nickel agarose affinity chromatography column, washing and eluting to finally obtain the SPA and HIV gp41 fusion protein.