CN118393137B - Detection kit for intestinal tract protective bacteria and application thereof - Google Patents
Detection kit for intestinal tract protective bacteria and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology detection, and particularly relates to a detection kit for intestinal tract protective bacteria and application thereof. The detection kit comprises a detection antibody 5H4, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is selected from SEQ ID NO. 2; the amino acid sequence of the light chain variable region is selected from SEQ ID NO. 6. The antibody can rapidly, sensitively and accurately detect the S-layer protein of the lactobacillus, and the detection kit prepared by the antibody can realize more sensitive, high-specificity and rapid detection of the lactobacillus, eliminates false positive interference caused by cross reaction and has good application prospect.
Description
Technical Field
The invention belongs to the field of biotechnology detection, and particularly relates to a detection kit for intestinal tract protective bacteria and application thereof.
Background
Lactobacillus (Lactobacillus) is a common intestinal probiotic. Lactobacillus is an important component of many health foods and beverages, including yogurt, fermented milk, and other probiotic products. Lactobacillus has multiple positive effects on human health, it can help to break down food, especially lactose, which is helpful to many people because they cannot digest lactose naturally, and in addition, lactobacillus can stimulate and enhance our immune system, keep intestinal microorganisms balanced, and in addition lactobacillus can inhibit the growth of harmful bacteria by competing for nutrients and living space. Lactobacillus is widely used as a probiotic, and increasing intake in the daily diet can help improve intestinal health.
S-layer proteins are Surface proteins found in many bacteria and archaea, forming a regular and very uniform structure, known as the S-layer. "S" herein stands for "Surface". The S-layer is usually composed of one or more framework protein subunits which self-assemble to form an acid-resistant and heat-resistant two-dimensional crystal array, and the acid-resistant and heat-resistant two-dimensional crystal array is covered on the surface of a thallus.
Monoclonal antibodies refer to antibodies raised from a single B cell clone that are highly homogeneous and directed against only a particular epitope. The hybridoma (hybridoma) antibody technology is generally prepared by adopting a hybridoma technology, and is based on a cell fusion technology, sensitized B cells with the capability of secreting specific antibodies and myeloma cells with the capability of proliferation are fused into B cell hybridomas. By culturing a cell population with a single hybridoma cell having such characteristics, a monoclonal antibody, which is a specific antibody against an epitope, can be produced. In view of the homogeneous nature, high specificity and high affinity of monoclonal antibodies, genetically engineered antibodies have been widely used in the prevention, diagnosis, treatment and purification of diseases.
The detection and judgment method for Lactobacillus mainly comprises the following steps:
and (3) observing by a microscope: this method is commonly used for the preliminary identification of microorganisms. It is based on the shape, size and staining reaction of the microorganisms. The method has the advantages of simplicity, convenience and rapidness, and has the defects that the strain level cannot be identified, and generally, only large categories can be distinguished;
Culture counting method: this method is commonly used for measuring the bacterial count. The advantage is that information on the size, shape and color of bacterial colonies can be visually seen, but the disadvantage is that specific culture conditions are required and that microorganisms that cannot grow on a specific medium cannot be detected.
Molecular biology method: such as PCR (polymerase chain reaction), microbiota fingerprint analysis (e.g., DGGE, TGGE), high throughput sequencing, and the like. The method has the advantages of high accuracy and reproducibility, and can identify the level of strains and even strains. However, the disadvantages are high technical requirements, high cost, and the need for corresponding equipment and software for data analysis, and the reaction affinity is greatly reduced compared with monoclonal antibodies.
Disclosure of Invention
The invention aims to overcome the defects of the various lactobacillus detection methods and provides a detection kit for the intestinal tract protective bacteria, which has the advantages of strong specificity, high sensitivity, rapidness and high efficiency and application thereof.
In order to achieve the above purpose, the invention provides a detection kit for intestinal tract protection bacteria, which comprises a capture antibody, a coating solution, an eluent and a detection antibody.
Preferably, the capture antibody is monoclonal antibody 5H4, and the detection antibody is horseradish peroxidase-labeled anti-mouse IgG Fc antibody.
Preferably, the monoclonal antibody 5H4 consists of a heavy chain variable region as shown in SEQ ID NO. 2 and a light chain variable region as shown in SEQ ID NO. 6.
Preferably, the heavy chain variable region consists of HCDR1 as shown in SEQ ID NO. 3, HCDR2 as shown in SEQ ID NO. 4 and HCDR3 as shown in SEQ ID NO. 5; the light chain variable region consists of LCDR1 shown as SEQ ID NO. 7, LCDR2 shown as SEQ ID NO. 8 and LCDR3 shown as SEQ ID NO. 9.
In another preferred embodiment of the present invention, there is also provided a detection antibody 5H4 against the S-layer protein monoclonal antibody.
Preferably, the monoclonal antibody 5H4 consists of a heavy chain variable region as shown in SEQ ID NO. 2 and a light chain variable region as shown in SEQ ID NO. 6.
Preferably, the heavy chain variable region consists of HCDR1 as shown in SEQ ID NO. 3, HCDR2 as shown in SEQ ID NO. 4 and HCDR3 as shown in SEQ ID NO. 5; the light chain variable region consists of LCDR1 shown as SEQ ID NO. 7, LCDR2 shown as SEQ ID NO. 8 and LCDR3 shown as SEQ ID NO. 9.
In another preferred embodiment of the present invention, a method for specifically, rapidly and simply detecting lactobacillus of intestinal tract protecting bacteria in a sample is provided, and a glass plate agglutination test is performed on a sample to be detected by using a reagent containing monoclonal antibody 5H 4.
Preferably, in the preferred embodiment of the present invention, a detection kit and an application of the detection antibody of the anti-S-layer protein monoclonal antibody in detecting lactobacillus of intestinal tract protection bacteria are also provided.
The technical scheme of the invention has the following effects:
The invention prepares the detection antibody 5H4 of the anti-S-layer protein monoclonal antibody, which can specifically identify the S-layer protein of the lactobacillus of the intestinal tract protective bacteria, wherein the monoclonal antibody 5H4 comprises a heavy chain variable region shown as SEQ ID NO. 2 and a light chain variable region shown as SEQ ID NO. 6.
The lactobacillus double-antibody sandwich detection method or the detection kit based on the detection antibody 5H4 provided by the invention has the effects of high sensitivity, good stability and strong specificity, and can detect a large number of samples simultaneously.
The method for rapidly and simply detecting the intestinal tract protective bacteria lactobacillus in the sample based on the specificity of the detection antibody 5H4 provided by the invention is that the sample to be detected is subjected to the glass plate agglutination test detection by using the reagent containing the monoclonal antibody 5H4, and the method has the detection effect of rapidness, sensitivity and high distinguishing degree, and has no cross reactivity with bifidobacteria, acidophilus, butyric acid bacillus, escherichia coli and enterococcus faecium in the detection process.
Drawings
Fig. 1: SDS-PAGE analysis of Lact-SLP antigen fragments.
Fig. 2: SDS-PAGE analysis of monoclonal antibody 5H 4.
Fig. 3: detection titers of monoclonal antibody 5H 4.
Fig. 4: and (3) carrying out sandwich detection correlation analysis on lactobacillus double-antibody based on monoclonal antibody 5H 4.
Detailed Description
While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Example 1: preparation of monoclonal antibody against Lact-SLP
Commercial ATCC8008 Lactobacillus sp standard strain is purchased, the external S-Layer Protein (SLP) gene of Lactobacillus is taken as a template, a gene fragment of the Lact-SLP antigen polypeptide is obtained through PCR reaction, and a Lact-SLP recombinant plasmid is constructed, and is subjected to double enzyme digestion with a pET-32a (+) vector, after the purification of the Lact-SLP antigen fragment and the pET-32a (+) are connected, the pET-32a (+) is provided with an HIS tag, and the pET-32a (+) -Lact-SLP recombinant plasmid is constructed and is transferred into DH5 alpha competence to pick up monoclonal delivery measurement.
Lact-SLP antigen fragment:
SKNSNVKFAGADGKFADTVKVELGQNGTLTTPISVQVSNVNALDLSNANGVNFYNASNGSQVTKGSVNVTAGLIGRLNVSTVASEILKNCAAYQVSNGKPVSQLPDQKAVVADVNAALKAANIPVDNAGWFTAPISLSVNVKASSSINGVGCYFTCTVNVANGKDMTVPSQSKTIMHNAYYYDKDAKRVGTDKLTRYNSVTVAMNTTTINGKAYYEVIENGKATGKFINADNIDGTK（SEQ ID NO:1）.
Transferring the pET-32a (+) -Lact-SLP recombinant plasmid with correct sequence into BL21 (DE 3) competent cells, picking positive monoclonal into LB culture medium containing 100 mg/L ampicillin, culturing until OD600 is 0.6-0.8, inducing overnight with 1 mmol/L IPTG at 25 ℃ and sampling, performing expansion culture, taking induced thalli, and performing ultrasonic crushing until liquid is clear. The supernatant was subjected to a column treatment with Ni-Agarose Resin and analyzed by SDS-PAGE containing 200 mmol/L imidazole-absorption Buffer eluting Lact-SLP, lact-SLP antigen fragments, wherein lane M: protein markers; lane 1: supernatant of the induced thalli; lane 2: precipitation of the thalli after induction; lanes 3-5: washing impurity liquid; lanes 6-8: eluent (see FIG. 1).
The results of FIG. 1 show that the Lact-SLP antigen fragment successfully induces expression, and the molecular weight is the same as the expected value, and the purity is more than 90 percent, so that the Lact-SLP antigen fragment can be used for subsequent mouse immunization.
Loading the purified Lact-SLP antigen fragment into a dialysis belt and dialyzing with PBS; a6-8 week old female, BALB/c mouse was immunized with purified Lact-SLP. The first immunization is fully emulsified by mixing Lact-SLP (100 mug) with Freund's complete adjuvant according to 1:1, and injected subcutaneously; collecting blood from tail vein of 1 week after exemption, continuously immunizing for 3 times, recovering SP2/0 cells 2 weeks before fusion, performing expansion culture with RPMI-1640 complete medium containing 10% fetal bovine serum, changing liquid with RPMI-1640 complete medium containing 20% fetal bovine serum 1 d before cell fusion, mixing collected spleen cells and SP2/0 cells according to the ratio of 8:1, and performing cell fusion with polyethylene glycol cell fusion reagent at 37 ℃ to obtain hybridoma cell strain 5H4; hybridoma cell line 5H4 was injected into the abdominal cavity of 8-10 week old BALB/c female mice pretreated with liquid paraffin in an amount of 1X 10 6/mouse, ascites were extracted when the abdomen of the mice swelled after 10-14 days of feeding observation, monoclonal antibody 5H4 was purified by affinity chromatography Protein G Sepharose Fast Flow, and purity of the monoclonal antibody was measured by SDS-PAGE and reached more than 90% (see FIG. 2).
The results in FIG. 2 show that the monoclonal antibody 5H4 is treated with the reducing agent to form two bands of heavy chain and light chain, and the protein electrophoresis pattern accords with the structural characteristics of the monoclonal antibody.
Example 2: identification of anti-Lact-SLP monoclonal antibody 5H4
2.1 Identification of antibody subtypes: subtype of hybridoma cell strain was identified using the mouse monoclonal antibody subtype identification kit from Roche company, subtype of 5H4 antibody was IgG1 type, and light chain was kappa chain.
2.2 Determination of antibody variable region sequences: extracting mRNA of hybridoma cells, reversely transcribing the mRNA into cDNA, performing high-fidelity PCR amplification by using a variable region universal primer, inserting a PCR amplified fragment into a T carrier for DNA sequence determination, and translating the DNA sequence determination result: the amino acid sequence of the heavy chain variable region of the coded monoclonal antibody 5H4 is shown as SEQ ID No:2, the amino acid sequence of the light chain variable region is shown as SEQ ID No: shown at 6.
Heavy chain variable region of 5H 4:
LRLSCAASRFTFSNKDNSQNMLYLQMNSGSGGSSYYADSVKGYGYYGMDVWGQGTXVTVSSPVTPGEPASISLRAEDTAVVWGQGTAPGKGLEWSLLHSNGYNYLDWYLQKPGEAEDVGVF（SEQ ID No：2）;
The heavy chain variable region of 5H4 consists of HCDR1, HCDR2 and HCDR 3;
HCDR1：RFTFSNKDN（SEQ ID No：3）；
HCDR2：SSYYADSVKGYGYYGMDVWG（SEQ ID No：4）；
HCDR3：LRAEDTAVVWGQGTA（SEQ ID No：5）；
light chain variable region of 5H 4:
SQSLLHSNGYNYLDWYLQKPGQSPTPLSLPVTPGEPASISCRSSQMQALQTPYTFGQGTVPDRFSGSGSGTDDWKYGYYGMDVCCLVLLTGVRAQVQLVESGGGLVQPGGSLRGLVQPGGSLRLS（SEQ ID No：6）;
The light chain variable region of 5H4 consists of LCDR1, LCDR2 and LCDR 3;
LCDR1：GYNYLDWY（SEQ ID No：7）；
LCDR2：GEPASISCRSSQMQALQ（SEQ ID No：8）；
LCDR3：SGTDDWKYGYYGM（SEQ ID No：9）；
2.3 potency detection of monoclonal antibody 5H 4: the coated Lact-SLP antigen fragment was serially diluted with a coating buffer to coat a 96-well ELISA plate at 100. Mu.L/well, and cooled overnight at 4 ℃. The enzyme label plate is taken out the next day and returned to room temperature, 200 mu L of PBST solution is injected into each hole, the shaking table is oscillated for 3 min, the washing liquid is thrown off forcefully, the washing liquid is taken out on the absorbent paper, and the washing is continued for 2 times. The following washing method was the same. After sufficient washing, the ELISA plate was blocked with blocking buffer, 200. Mu.L/well, incubated in an incubator at 37℃for 2 h and then removed for baking. Monoclonal antibody 5H4 was serially diluted to correspond to the first 7 rows and columns of the ELISA plate, negative control was added to row 8, 100. Mu.L/well, incubated at 37℃for 1H, washed and dried. 100 μl of HRP-labeled goat anti-mouse IgG diluted 1:3000 was added to each well, incubated at 37 ℃ for 1 h, and washed and patted dry. 100. Mu.L of a color-developing solution (TMB to substrate solution ratio: 1:5) was added to each well, and the mixture was reacted at 37℃in the dark for 15 min, and 100. Mu.L of a stop solution (2 mol/L sulfuric acid) was added to each well after removal, and the absorbance A450 was measured by using a microplate reader (see FIG. 3).
The results in FIG. 3 show that the titer of monoclonal antibody 5H4 can reach 1:640000, indicating that the sensitivity of monoclonal antibody 5H4 is high.
Example 3: lactobacillus double-antibody sandwich detection
The ELISA plate was coated with 10. Mu.g/mL monoclonal antibody 5H4, 100. Mu.L/well, overnight at 4 ℃; washing the reaction plate three times with PBST for 3min each time, 200 mu L/hole, and spin-drying the reaction plate; CBS with 0.2% gelatin, 200. Mu.L/well, blocked for 2h at 37 ℃; repeating the above steps, diluting ATCC8008 Lactobacillus sp with PBST to 2.23×105、3.67×104、2.54×103、6.48×103、2.46×102、4.82×101cfu/mL series of concentration, and setting a PBST blank; mu.L of sample was added to each well and incubated lh at 37 ℃. Washing the reaction plate three times with PBST for 3min each time, 200 mu L/hole, and spin-drying the reaction plate; adding enzyme-labeled antibody 5H4-HRP (horseradish peroxidase (HRP) labeled 5H4,2 mu g/mL), 100 mu L/well, and 37: 37C for 1H reaction; washing the reaction plate three times with PBST for 3min each time, 200 mu L/hole, and spin-drying the reaction plate; adding 100 mu L/hole of a substrate TMB, and developing for 10min; stop solution was added at 50. Mu.L/well. OD450nm was detected with a microplate reader (FIG. 4).
The results of fig. 4 show that: the detection range of the lactobacillus antigen detection obtained by the invention is 10 3～104 cfu/mL, and the correlation R2=0.99.
Example 4: specificity detection result of anti-Lact-SLP monoclonal antibody 5H4 on different strains
The standard substances of lactobacillus, bifidobacterium, acidophilus, butyric acid bacillus, escherichia coli and enterococcus faecium are respectively used for carrying out a glass plate agglutination test with the monoclonal antibody 5H4 screened, a small number of colonies of the lactobacillus, bifidobacterium, acidophilus, butyric acid bacillus, escherichia coli and enterococcus faecium selected on a flat plate are dipped by an inoculating loop, evenly mixed in physiological saline, then respectively dripped on a clean glass slide marked in advance, and meanwhile, the monoclonal antibody 5H4 is dripped, and a control group of the lactobacillus, bifidobacterium, acidophilus, butyric acid bacillus, escherichia coli, enterococcus faecium, physiological saline and monoclonal antibody and physiological saline is arranged. The results are shown in Table 1, and the results show that the monoclonal antibody 5H4 has good specificity, only reacts positively with lactobacillus, and is negative with bifidobacterium, acidophilus, butyric acid bacillus, escherichia coli and enterococcus faecium.
Table 1 monoclonal antibody 5H4 specificity identification
Positive results of the glass plate agglutination test are shown in +A; negative for the glass plate agglutination test results.
The results in Table 1 show that the monoclonal antibody 5H4 against Lactobacillus can specifically detect Lactobacillus; has no cross-reactivity with bifidobacterium, acidophilus, butyric acid bacillus, escherichia coli and enterococcus faecium.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (3)
1. The detection kit for the intestinal tract protective bacteria is characterized by comprising a detection antibody, wherein the intestinal tract protective bacteria are lactobacillus, and the detection antibody is a monoclonal antibody consisting of a heavy chain variable region shown as SEQ ID NO. 2 and a light chain variable region shown as SEQ ID NO. 6.
2. The test kit according to claim 1, wherein the heavy chain variable region consists of HCDR1 as shown in SEQ ID No. 3, HCDR2 as shown in SEQ ID No. 4 and HCDR3 as shown in SEQ ID No. 5; the light chain variable region consists of LCDR1 shown as SEQ ID NO. 7, LCDR2 shown as SEQ ID NO. 8 and LCDR3 shown as SEQ ID NO. 9.
3. Use of a detection kit according to any one of claims 1-2 for the preparation of a product for detecting lactobacillus as an intestinal protective bacterium.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002361215A1 (en) * | 2001-12-28 | 2003-07-15 | Nederlandse Organisatie Voor Toegepastnatuurwetenschappelijk Onderzoek Tno | Modified bacterial surface layer proteins |
| CN101638660A (en) * | 2008-08-01 | 2010-02-03 | 长春理工大学 | Construction of lactobacillus acidophilus S-layer protein surface display system |
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| AU2014312456B2 (en) * | 2013-08-30 | 2017-07-06 | Aprilbio Co., Ltd | An anti serum albumin Fab-effector moiety fusion construct, and the preparing method thereof |
| WO2016014565A2 (en) * | 2014-07-21 | 2016-01-28 | Novartis Ag | Treatment of cancer using humanized anti-bcma chimeric antigen receptor |
| KR102206392B1 (en) * | 2017-05-08 | 2021-01-21 | 고려대학교 산학협력단 | Multiple detection of bacterial pathogens through cell wall binding domain complexes |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002361215A1 (en) * | 2001-12-28 | 2003-07-15 | Nederlandse Organisatie Voor Toegepastnatuurwetenschappelijk Onderzoek Tno | Modified bacterial surface layer proteins |
| CN101638660A (en) * | 2008-08-01 | 2010-02-03 | 长春理工大学 | Construction of lactobacillus acidophilus S-layer protein surface display system |
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