CN108982875A

CN108982875A - The preparation and application of Selenoprotein P colloidal-gold detecting-card

Info

Publication number: CN108982875A
Application number: CN201810915850.2A
Authority: CN
Inventors: 张明程; 杨建国
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-08-13
Filing date: 2018-08-13
Publication date: 2018-12-11

Abstract

The invention discloses the preparation and application of Selenoprotein P colloidal-gold detecting-card.Selenoprotein P colloidal-gold detecting-card disclosed by the invention, including connection gasket interconnected, absorption pad and coated film with the detection line and nature controlling line that are separated from each other, coated film is between the connection gasket and connection gasket, and load has colloid gold label SePm antibody on connection gasket；Detection line is coated with SePm antibody, and quality inspection line is coated with the SePm albumen as antigen；Colloid gold label SePm antibody is to modify the compound that SePm antibody obtains using colloidal gold；SePm antibody is the monoclonal antibody obtained using SePm albumen as antigen or polyclonal antibody；SePm albumen is the protein that amino acid sequence is sequence 2.It is demonstrated experimentally that Selenoprotein P colloidal-gold detecting-card of the invention can be used to detect Selenoprotein P, may further be used to evaluate human body whether selenium deficiency.

Description

The preparation and application of Selenoprotein P colloidal-gold detecting-card

Technical field

The present invention relates in field of biotechnology, the preparation and application of Selenoprotein P colloidal-gold detecting-card.

Background technique

Selenium (Selenium, Se) is one of microelement necessary to mammal.There are many physiological function, selenium deficiency to lead for selenium Cause a variety of pathological states, such as: hepatonecrosis, cardiomyopathy, cardiovascular disease initiation potential degree increase, the death rate of aids patient increase High, epidemiological survey shows that selenium-supply can reduce the disease incidence of cancer.Famous is long-lived without cancer village Bama of Guangxi, local Water and soil Se content are higher than other ten times of areas.China is the country of one 2/3 regional selenium deficiency, and many people are in selenium deficiency shape State, therefore the health status of people can be improved in supply Se scientific.

Selenium is to play its physiological function by selenoprotein (Selenoprotein).Selenoprotein is a kind of special egg White matter, it is characterized in that the selenium in albumen exists in the form of selenocysteine (selenocysteine, SeCys).SeCys is big Multidigit plays an important role to the structure and function of albumen in the activated centre of albumen.SeCys is by a special password Son --- (UGA is as terminator codon in general protein to encode for the UGA codon in opening code-reading frame (ORF) Work), it is incorporated into the polypeptide chain synthesized while translation, therefore SeCys is regarded as constitutive protein matter now 21st kind of amino acid.

So far, in mammals it has been found that 25 kinds of selenoproteins, including 4 kinds of glutathione peroxidases, 3 kinds of de- iodine Enzyme, thioredoxin reductase etc. and the unknown selenoprotein of other function, such as Selenoprotein P, selenoprotein W, 15kDa selenoprotein.

Selenoprotein P (Selenoprotein P, SelP) is initially to be found in rat plasma in 1977 by Herrman, Later Burk and Tappel also demonstrates this albumen, because being the selenoprotein found in blood plasma, just with English first word of blood plasma Female P name, this title of Selenoprotein P, SelP are received by everybody always.

Selenoprotein P content in blood plasma is very low, therefore purifies and separates are highly difficult.After repeatedly failing, China in 1987 Scholar Yang Jianguo uses immunoaffinity chromatography technology in the laboratory of U.S. Burk finally, has isolated and purified rat for the first time in the world Blood plasma Selenoprotein P.The partial biochemical characteristics for having studied the albumen again later, further research and develop and have cloned rat and the selenium egg of people The gene of white P.

Selenoprotein P is widely distributed in the tissue.SelP is different from other selenoprotein in structure, and general selenoprotein contains only one A SeCys, and SelP contain at least ten SeCys (people, mouse, rat have 10, ox have 12, grouper have 17).People Selenoprotein P (HSelP) cDNA overall length 2048bp, encodes 381 amino acid, containing TGA in 10 reading frames, encodes 10 half Guangs of selenium Propylhomoserin, 3 ' non-translational regions are conservative, and it to be synthesis that there are two SECIS (Selenocysteine insertion sequence) The essential structure of HSelP can instruct multiple selenocysteines to mix.General selenoprotein only has a SECIS, and SelP There are 2 SECIS, and it is strongest in all selenoproteins that it, which instructs SeCys incorporation effect,.In addition, Selenoprotein P contains multiple groups The basic amino acids such as propylhomoserin, lysine form two histidine enrichment regions, have the characteristic of heparin-binding and can utilize this area Characteristic carries out protein purification.60% selenium is present in Selenoprotein P in human blood, remaining for glutathione peroxidase and its His selenoprotein.

10 SeCys that HSelP contains, wherein first SeCys is located at the 40th of N-terminal, remaining 9 are located at C-terminal 1/3.HSelP naturally occurring two hypotypes in blood plasma, a kind of Selenoprotein P for overall length, one kind are the shorter hypotype of Selenoprotein P, Albumen synthesis terminates in advance i.e. at the 2nd SeCys/UGA.The N-terminal of two kinds of hypotypes is identical, and C-terminal is different.Contain up to 10 The recombinant expression of the human selenoprotein P of SeCys is very difficult, and SelP is from the purification steps troublesome in blood plasma, and yield is lower, keeps It is active difficult, because it is difficult to have obtained a large amount of albumen, since the seventies finds SelP, characteristic and function are still unclear so far Chu.

Document report, when making one blood selenium content and reaching saturation, it is micro- that glutathione peroxidase needs to take selenium element 35 Gram/day, and blood Selenoprotein P then needs to take 49 micro- grams/day.Therefore, scientist has been recognized that Selenoprotein P can be as human body selenium The index of state.Evaluate human body whether selenium deficiency, measure Selenoprotein P content.For this purpose, needing a kind of quickly detection selenium egg at present The Method and kit for of white P content.

Summary of the invention

The technical problem to be solved by the present invention is to how detect Selenoprotein P and Se content.

In order to solve the above technical problems, present invention firstly provides a kind of immune detection product of Selenoprotein P, the product Including connection gasket interconnected, absorption pad and coated film with the detection line and nature controlling line that are separated from each other, the coated film Between the connection gasket and the absorption pad, it is characterised in that: load has colloid gold label SePm anti-on the connection gasket Body；The detection line is coated with SePm antibody, and the quality inspection line is coated with the SePm albumen as antigen；

The colloid gold label SePm antibody is to modify the compound that SePm antibody obtains using colloidal gold；

The SePm antibody is the monoclonal antibody obtained using the SePm albumen as antigen or polyclonal antibody；

The SePm albumen is following A1), A2) or A3):

A1) amino acid sequence is the protein of sequence 2；

A2) amino acid sequence of sequence 2 in sequence table is passed through to the substitution and/or missing of one or several amino acid residues And/or addition and protein with the same function；

A3) in A1) or the obtained fused protein of N-terminal A2) or/and C-terminal connection label；

The monoclonal antibody is the monoclonal antibody of hybridoma secretion, and the hybridoma is by the SePm The cell that the splenocyte of the immune animal obtained after protein immune animal is merged with myeloma cell；

The polyclonal antibody is the polyclonal antibody obtained using animal described in the SePm protein immunization.

The SePm antibody can SePm albumen described in specific recognition.

In the said goods, the coated film can be fine for the nitric acid with the shown detection line and the nature controlling line being separated from each other Tie up plain film.

In the said goods, the item number of the detection line can be 1-5 item.

The product can also contain sample pad, the sample pad be located at the non-coated film side of the connection gasket and with it is described Connection gasket is connected.

In the said goods, the package amount of the SePm antibody can be 2 μ g/cm in the detection line.

The package amount of the SePm antigen can be 0.2 μ g/cm on the quality inspection line.

In order to make A1) in protein convenient for purifying, amino acid sequence shown in sequence 2 can be formed in by sequence table The upper label as shown in the table of amino terminal or carboxyl terminal connection of protein.

Table: the sequence of label

Label	Residue	Sequence
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned A2) in SePm albumen, to have 75% or 75% or more with the amino acid sequence of protein shown in sequence 2 Identity and protein with the same function.Described to have 75% or 75% or more identity be with 75%, have 80%, With 85%, with 90%, with 95%, with 96%, with 97%, with 98% or with 99% identity.

Above-mentioned A2) in SePm albumen can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain It arrives.

Above-mentioned A2) in the encoding gene of SePm albumen can be by will be in DNA sequence dna shown in 8-1111 of sequence 1 The codon of one or several amino acid residues is lacked, and/or carries out the missense mutation of one or several base-pairs, and/or The coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table obtains.Wherein, shown in 8-1111 of sequence 1 SePm albumen shown in DNA molecular coded sequence 2.

In the said goods, the preparation method of the SePm albumen can include: import the encoding gene of the SePm albumen In microorganism, recombinant microorganism is obtained, expresses the encoding gene of SePm albumen described in the recombinant microorganism, is obtained described SePm albumen.

In the said goods, the encoding gene of the SePm albumen can be b1), b2), b3) or b4):

B1) cDNA molecule or DNA molecular shown in 8-1111 of sequence 1 in sequence table；

B2) coded sequence is 8-1111 DNA moleculars of sequence 1 in sequence table；

B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% or more identity, and encodes the SePm The cDNA molecule or DNA molecular of albumen；

B4) under strict conditions with b1) or b2) or b3) nucleotide sequence hybridization that limits, and encode the SePm albumen CDNA molecule or DNA molecular.

Wherein, the encoding gene can be DNA, such as cDNA, genomic DNA or recombinant DNA.

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of coding SePm albumen of the invention.Those have and the present invention by manually modified The nucleotide sequence 75% of isolated SePm albumen or the nucleotide of higher identity, as long as coding SePm albumen and tool There is SePm protein function, is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.

Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair Amino acid sequence shown in bright coded sequence 2 composition protein nucleotide sequence have 75% or higher or 85% or Higher or 90% or higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or computer software It is evaluated.Using computer software, identity between two or more sequences can be indicated with percentage (%), can be with For evaluating the identity between correlated series.

In above-mentioned application, the stringent condition can be as follows: 50 DEG C, in 7% lauryl sodium sulfate (SDS), 0.5M NaPO₄Hybridize in the mixed solution of 1mM EDTA, is rinsed in 50 DEG C, 2 × SSC, 0.1%SDS；May be used also are as follows: 50 DEG C, 7%SDS, 0.5M NaPO₄Hybridize in the mixed solution of 1mM EDTA, is rinsed in 50 DEG C, 1 × SSC, 0.1%SDS；May be used also Are as follows: 50 DEG C, in 7%SDS, 0.5M NaPO₄Hybridize in the mixed solution of 1mM EDTA, at 50 DEG C, 0.5 × SSC, 0.1% It is rinsed in SDS；May be used also are as follows: 50 DEG C, in 7%SDS, 0.5M NaPO₄Hybridize in the mixed solution of 1mM EDTA, at 50 DEG C, It is rinsed in 0.1 × SSC, 0.1%SDS；May be used also are as follows: 50 DEG C, in 7%SDS, 0.5M NaPO₄In the mixed solution of 1mM EDTA Hybridization, rinses in 65 DEG C, 0.1 × SSC, 0.1%SDS；It can also are as follows: in 6 × SSC, the solution of 0.5%SDS, at 65 DEG C Hybridization, then with 2 × SSC, 0.1%SDS and 1 × SSC, it is primary that 0.1%SDS respectively washes film；It can also are as follows: 2 × SSC, 0.1%SDS Solution in, hybridize at 68 DEG C and wash film 2 times, each 5min, and in 0.5 × SSC, the solution of 0.1%SDS, at 68 DEG C Lower hybridization simultaneously washes film 2 times, each 15min；Can also are as follows: 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution in, 65 DEG C Under the conditions of hybridize and wash film.

Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

In above-mentioned application, B2) described in the nucleic acid molecules containing coding SePm protein expression cassette (SePm gene expression Box), it is the DNA for referring to express SePm protein in host cell, which not only may include starting SePm genetic transcription Promoter may also include the terminator for terminating SePm genetic transcription.Further, the expression cassette may also include enhancer sequence.

The recombinant microorganism can be that pET-SePm is imported recombinant microorganism obtained in the microorganism, the pET- SePm is by the multiple cloning sites of the importing pET-30aSETB carrier of DNA molecular shown in 8-1111 of sequence 1 in sequence table Between the obtained recombinant vector that can express the fusion protein that the SePm or SePm and other polypeptides are formed.

In the said goods, the preparation method of the colloid gold label SePm antibody can include: by colloidal gold and the SePm Antibody is reacted to obtain the colloid gold label SePm antibody.

The proportion of the colloidal gold and the SePm antibody can be colloidal gold described in 10ng: SePm antibody described in 1 μ g.

The diameter of the colloidal gold can be 15-40nm.

In the said goods, the animal can be for 1), 2) or 3):

1) mammal；

2) rabbit；

3) mouse.

The SePm albumen, the encoding gene of the SePm albumen, the SePm antibody or the hybridoma also belong to In protection scope of the present invention.

The present invention also provides detection or the methods of auxiliary detection Selenoprotein P, which comprises adds to sample to be tested In the sample pad of the product, whether it is presented whether red determining sample to be tested contains selenoprotein according to the detection line of the product P: if the detection line of the product takes on a red color with quality inspection line, the sample to be tested contains or candidate contains Selenoprotein P；As described in The detection line of product does not take on a red color, and quality inspection line takes on a red color, and the sample to be tested does not contain or candidate is without containing Selenoprotein P；As institute The detection line and quality inspection line for stating product do not take on a red color, and whether the product failure, the sample to be tested contains Selenoprotein P not Know.

The present invention also provides following X1 or X2:

X1, the SePm albumen, the encoding gene of the SePm albumen, the SePm antibody, the hybridoma or Application during the product is any in following X1a-X1f:

X1a, preparation detection or auxiliary detection Selenoprotein P product；

X1b, detection or auxiliary detection Selenoprotein P；

X1c, preparation detection or auxiliary detection selenium product；

X1d, detection or auxiliary detection selenium；

X1e, preparation evaluation human body whether selenium deficiency product；

X1f, evaluation human body whether selenium deficiency；

Application of the method for X2, the detection or auxiliary detection Selenoprotein P in above-mentioned X1d or X1f.

It is demonstrated experimentally that the immune detection product of Selenoprotein P of the invention can be used to detect Selenoprotein P, it may further For evaluate human body whether selenium deficiency.

Detailed description of the invention

Fig. 1 is the electrophoretogram of SePm expressing fusion protein after purification.Wherein, swimming lane 1- swimming lane 4 is the pure of different applied sample amounts SePm fusion protein after change.

Fig. 2 is the mostly anti-sensitivity of Dot hybridization SePm.Wherein, 1 indicate that the concentration of SePm fusion protein is 200 μ g/ Ml, 2 indicate that the concentration of SePm fusion protein is 20 μ g/ml, and 3 indicate that the concentration of SePm fusion protein is 2 μ g/ml, and 4 indicate SePm The concentration of fusion protein is 200ng/ml, and 5 indicate that the concentration of SePm fusion protein is 20ng/ml.Fig. 3 is to detect liver using SePm Selenoprotein P in cancerous cell line HepG2.Wherein 1 and 2 be training that liver cancer cell lines HepG2 free serum culture is collected after 24 hours Nutrient solution supernatant.

Fig. 4 is the testing result of SePm monoclonal antibody sensitivity.1:200ng/mL；2:20ng/mL；3:2ng/mL；4:0.2ng/ mL。

Fig. 5 is the testing result of SePm monoclonal antibody specificity.M is protein molecular weight standard, and 1,2 and 3 be normal person's blood Clearly；4 be SePm fusion protein.

Fig. 6 is Selenoprotein P colloidal-gold detecting-card schematic diagram.

Specific embodiment

The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.

Experimental method in following embodiments is unless otherwise specified conventional method.

Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified.

Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.

The preparation of embodiment 1, SePm and its antibody

According to the human selenoprotein P gene order (NM_005410 sequence) of Genbank, by selenocysteine codon TGA Cystein codons TGT (or TGC) is sported, (codon uses frequency in conjunction with bacillus coli gene codon-bias feature Rate), codon is optimized, the encoding gene of human selenoprotein P mutein (being named as SePm) is obtained, SePm is compiled The sequence of code gene is as shown in 8-1111 of sequence 1 in sequence table, SePm shown in sequence 2 in polynucleotide.

One, the preparation of SePm

DNA molecular shown in artificial synthesized sequence 1,2-7 of sequence 1 are the identification sequence of NheI, 1112-1117 Position is the identification sequence of XhoI.Using NheI and XhoI to DNA molecular double digestion shown in sequence 1, target gene fragment is obtained； Using NheI and XhoI to pET-30aSETB carrier (i.e. pET-30b (+), NOVAGEN company) double digestion, carrier framework is obtained. Target gene fragment and carrier framework are connected, recombinant vector is obtained, which is named as pET-SePm, pET-SePm energy Express the fused protein (hereinafter referred to as SePm fusion protein) of SePm fusion His label.

PET-SePm is imported into e. coli jm109 (DE3), recombinant bacterium is obtained, which is named as ZH2Y- SePm, ZH2Y-SePm can express SePm fusion protein.By pET-30aSETB vector introduction e. coli jm109 (DE3), obtain Control strain.

Prepare SePm fusion protein:

1, by ZH2Y-SePm strain inoculated into the LB culture medium of the kanamycin sulfate containing 50 μ g/ml, at 37 DEG C and 12h is cultivated in 200rpm shaking table.

2, add IPTG to its final concentration of 1mM inducible protein expression, protein induced expression 5 after the completion of step 1 in bacterium solution After hour, obtained bacterium solution is take out of the shaker, bacterium is collected by centrifugation, obtains bacterial precipitation.

3, after bacterial lysate being added in bacterial precipitation, ultrasonication bacterial cell, then centrifuging and taking supernatant.

4, the supernatant of step 3 nickel column (HiTrap Chelating prepacked column) is crossed to purify.To the purpose of elution Albumen carries out the identification of SDS-PAGE protein electrophoresis, as a result as shown in Figure 1.

It carries out the ZH2Y-SePm bacterial strain to shake bottle and cultivates and carry out SePm expressing fusion protein purifying, obtain after purification SePm fusion protein, SePm fusion protein yield after purification is about 10-30mg/L bacterium solution.

Fig. 1 shows that this method can be obtained with soluble, high yield SePm fusion protein.

Control strain is subjected to above-mentioned experiment, no purpose band occurs.

Two, SePm mostly anti-preparation

The SePm fusion protein of step 1 is dissolved in the SePm fusion for obtaining that SePm fusion protein concentration is 2mg/ml in PBS Protein solution.Using the SePm fusion protein of step 1 as immunogen immune rabbit (Jinan Jin Feng experimental animal Co., Ltd, newly Western blue rabbit), dissipate multiple intradermal injections.Reinforce primary immunization after every 4 weeks.Initial immunity Freund's complete adjuvant (complete Freund's adjuvant, CFA) mixed in equal volume with SePm fusion protein solution after be immunized rabbit, the dosage of SePm fusion protein For 100 micrograms；Immune multiple spot subcutaneous injection every time later.Paracentesis pericardii takes blood after 3 months, and 4 DEG C stand overnight.Next day is sucked out Faint yellow antiserum will be centrifuged 10 minutes under antiserum 2000rpm, remove blood clot, obtain the rabbit-anti people Duo Ke of SePm albumen Grand antibody (hereinafter referred to as SePm is mostly anti-).

It is detected using SePm fusion protein as antigen using how anti-SePm is, experimental result, which confirms that SePm is mostly anti-, to be known Other SePm fusion protein, and the SePm fusion protein (Fig. 2) that concentration is 20ng/ml can be recognized.

Utilize the Selenoprotein P of the how anti-detection liver cancer cell lines HepG2 of SePm, the results showed that, how anti-SePm is can identify liver cancer The Selenoprotein P (Fig. 3) of cell line HepG2.

Three, the preparation of SePm monoclonal antibody

Using the SePm fusion protein of step 1 as immunogen immune mouse, (Beijing China Fukang biotechnology share is limited Company, BALB/c, 9-12 week old) preparation SePm monoclonal antibody (i.e. SePm monoclonal antibody), the specific method is as follows:

1. mouse immune: being mice immunized with antigen with SePm fusion protein, be immunized and use intraperitoneal injection, first immunisation takes 100 microlitres of SePm fusion proteins (2mg/ml), are added 100 microlitres of physiological saline, then plus equivalent Freund's complete adjuvant, it is fully emulsified After be injected intraperitoneally, every volume injected carries out secondary immunity after being 50 microlitres, 2 weeks, mixed with antigen using incomplete Freund's adjuvant It closes and emulsifies, with for the first time, second of immune blood sampling in latter 14th day carries out Dot hybridization for immunization route and dosage.Two are used simultaneously Secondary immunization method booster immunization is primary, and interval takes blood to survey antibody after 2 weeks.

2. cell fusion: taking mouse of the serum titer higher than 104 after booster immunization, put to death, in sterile in superclean bench Spleen is taken out, carries out cell count after grinding, is centrifuged after splenocyte and myeloma cell SP20 are mixed in 8:1 ratio, 37 In DEG C water-bath by 800 microlitres of 50%PEG4000 of pre-temperature in the centrifuge tube that cell is added dropwise in 60 seconds, side edged is slight It shakes, after adding after 37 DEG C of water-bath standings act on 90 seconds, RPMI1640 culture solution is added to terminate reaction, then with 1000R/ Per minute, centrifugation 7 minutes (centrifugation radius 9CM), remove supernatant, and cell is resuspended with HAT selection culture solution, is added to by 100 microlitres/hole In the 96 hole microwell plates added with feeder layer (hybridoma method), culture plate is placed in 5%CO₂37 degree of lower trainings in incubator It supports.

3. the screening of positive colony and the monoclonal of positive cell: the fused 1st, changing within 7 days the mode of liquid with half amount Culture solution is replaced, after fusion 10 days, difference detection fusion cell supernatant antibody level, screening antibodies positive hole, after two days again It is rechecked, carries out first time monoclonal using limiting dilution assay, then carried out again second and third time is subcloned, to list After cloning obtains cell expansion culture, cell strain is put into -80 degree in 5 milliliters of centrifuge tubes and is saved.

4. the preparation and purification of odd contradictive hydroperitoneum: take 12 week old BALB female mices in intraperitoneal injection paraffin oil 0.5 milliliter every, one By the hybridoma of logarithmic growth phase (1-4) × 10 after week⁵A/milliliter injects mouse peritoneal, acquires ascites later, use is pungent Acid system purifies ascites monoclonal antibodies, obtains SePm monoclonal antibody.

5. the measurement of monoclonal antibody sensitivity:

Using antigen gradient dilution dot blot method on cellulose membrane, the sensitivity of monoclonal antibody is measured: by SePm antigen (SePm Fusion protein) gradient dilution is carried out, minimum to be diluted to 0.2ng/mL, SePm monoclonal antibody can detect the SePm antigen of various concentration (Fig. 4) illustrates SePm monoclonal antibody high sensitivity, has reached national testing requirements.

6. the measurement of monoclonal antibody specificity

Using the method for western-blot, the specificity of SePm monoclonal antibody is measured, sample used is the SePm fusion of purifying Albumen, normal person (not selenium deficiency individual) serum (3 blood serum samples), SePm monoclonal antibody can be with the natural selenium egg of specific recognition as the result is shown White P, specific good (Fig. 5).

The preparation and application of embodiment 2, Selenoprotein P colloidal-gold detecting-card

How anti-SePm monoclonal antibody and the SePm for being utilized respectively the preparation of embodiment 1 be as SePm Antibody preparation Selenoprotein P colloidal gold Detection card, specific steps are as follows:

One, colloid gold label Selenoprotein P antibody

The colloidal gold that diameter is 40nm is taken to make it combine in the buffer of pH 7.2 by stirring with SePm monoclonal antibody, colloid The proportion of gold and SePm monoclonal antibody is 10ng colloidal gold: 1 μ g SePm monoclonal antibody.Buffer used is 10mM Tris buffer.Using Supercentrifugal process removes unbonded SePm monoclonal antibody.In the SePm antibody that centrifugation bottom of the tube peony precipitating is colloid gold label Compound (colloidal gold-SePm antibody complex).

Two, colloidal gold-SePm antibody complex sprays gold pad

The precipitating that step 1 is obtained is resuspended with colloidal gold buffer (10mM Tris buffer), obtains compound suspension, Compound suspension is sprayed in gold pad, is dried in vacuo, the dosage of compound suspension is 2 μ l/cm in gold pad, obtains being coated with glue The gold pad of body gold-SePm antibody complex.

Three, the scribing line of immunochromatography film

Using SePm antibody as detection line (T line) coated antibody on nitrocellulose filter, with SePm fusion protein (SePm Antigen) as nature controlling line (C line) the coating object on nitrocellulose filter, the specific method is as follows:

SePm antibody-solutions (solvent PBS, solute are SePm antibody, and the concentration of SePm antibody is 2.0mg/ml) are utilized For the method for spraying in crossing (T line, detection line) on nitrocellulose filter, SePm antibody-solutions dosage is 1.0 μ l/cm.By SePm Antigenic solution (solvent PBS, solute be SePm fusion protein, SePm fusion protein concentration be 0.2mg/ml) spraying method in It crosses (C line, quality inspection line) on nitrocellulose filter, SePm antigenic solution dosage is 1.0 μ l/cm.Then will containing detection line and The nitrocellulose filter of quality inspection line is immunized after relative humidity is to carry out dehumidifier 4 hours under 10% drying condition below Chromatographic film, it is dry to be sealed for use.

Four, the preparation of detection card

Using plastic polyethylene plate as supporter (bottom plate), Selenoprotein P colloidal-gold detecting-card is assembled, according to sample to be tested Each section of flow direction detection card are as follows: the sample pad that is made of 3-5 layer filter paper is sprayed by one layer of folder among two layers of glass layer Be coated with the glass layer (connection gasket), immunochromatography film, absorption pad (Fig. 6) of colloidal gold-SePm antibody complex, connection gasket with Sample pad 3mm Chong Die with immunochromatography film, connection gasket and detection line close to but not be overlapped, sample pad 3mm Chong Die with connection gasket, Absorption pad 3mm Chong Die with immunochromatography film, absorption pad and quality inspection line close to but it is not be overlapped.It is cut into the strip of 5mm wide, is obtained Selenoprotein P colloidal-gold detecting-card will test and be named as Selenoprotein P colloidal-gold detecting-card A at line for the detection card of SePm monoclonal antibody, will It is named as Selenoprotein P colloidal-gold detecting-card B for the mostly anti-detection card of SePm at detection line, 4 DEG C of hermetically dryings save backup.

The anticipation reaction result of Selenoprotein P colloidal-gold detecting-card:

When measuring samples block through capillary action along detection by sample pad swimming horizontally forward, if test sample In contain Selenoprotein P, Selenoprotein P first in conjunction with the SePm antibody on colloidal gold-SePm antibody complex, formed antigen-antibody Compound, the compound continue to flow, and the antigen (Selenoprotein P) in antigen antibody complex is by preparatory coated SePm's herein Antibody capture is simultaneously retained down, and as red complex interception increases, a red line is gradually formed, without captured antigen Antibody complex continues flow forward, and the antibody in compound, with the antigen binding on quality inspection line, forms one at quality inspection line Red line.If be free of Selenoprotein P in test sample, colloidal gold-SePm antibody complex and the SePm being fixed on quality inspection line Antigen binding, and be enriched on quality inspection line, a red line is gradually formed, and red line cannot be formed at detection line.

When detecting sample to be tested, there is the positive reaction that is judged to of red stripes simultaneously in detection line and quality inspection line, it is to be checked Contain Selenoprotein P in sample；Only there is the negative reaction that is judged to of red stripes in quality inspection line, is free of Selenoprotein P in measuring samples； If quality inspection line does not occur red stripes, show detection card failure.

Five, the application of detection card

1) detection of urine sample

It selects the clinical urine sample for containing Selenoprotein P known to ten as sample to be tested, is utilized respectively Selenoprotein P colloid Gold detection card A and Selenoprotein P colloidal-gold detecting-card B is detected: urine sample being instilled Selenoprotein P colloidal-gold detecting-card respectively Sample pad on, observation is as a result, the results show that be the positive there are five the testing result of clinical urine sample after five minutes, there are five The testing result of clinical urine sample is feminine gender, Selenoprotein P colloidal-gold detecting-card A and Selenoprotein P colloidal-gold detecting-card B result one It causes, and consistent with clinical effectiveness, shows that two kinds of detection cards are used equally for the Selenoprotein P in detection urine, and accuracy rate is high.

2) detection of blood plasma (or serum) sample

It selects the clinical blood sample for containing Selenoprotein P known to ten as sample to be tested, is utilized respectively Selenoprotein P colloid Gold detection card A and Selenoprotein P colloidal-gold detecting-card B is detected: each sample takes 100 microlitres, and drop is examined in Selenoprotein P colloidal gold It surveys in the sample pad of card, observation has as a result, the results show that there is the testing result of eight clinical blood samples for the positive after five minutes The testing result of two clinical blood samples is feminine gender, and Selenoprotein P colloidal-gold detecting-card A and Selenoprotein P colloidal-gold detecting-card B are tied Fruit is consistent, and consistent with clinical effectiveness, shows that two kinds of detection cards are used equally for the Selenoprotein P in detection blood plasma, and accuracy rate It is high.

The above result shows that Selenoprotein P colloidal-gold detecting-card of the invention can be used to detect Selenoprotein P.

<110>Zhang Mingcheng, Yang Jianguo

<120>preparation and application of Selenoprotein P colloidal-gold detecting-card

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1118

<212> DNA

<213>artificial sequence

<400> 1

ggctagcggt ggtggtggta ctgaatctca agatcagagc tctctgtgca aacaaccgcc 60

agcatggagc attcgtgacc aagatccgat gctgaactcc aatggttccg taactgtcgt 120

tgcactgctg caggcaagct gttacctgtg cattctgcag gcatccaaac tggaagatct 180

gcgtgtcaaa ctgaagaaag aaggttacag caacatcagc tacattgtgg tcaaccatca 240

gggtatcagc tctcgtctga aatacactca tctgaagaac aaggtgtctg agcacattcc 300

ggtctatcag caagaagaga accagaccga tgtctggact ctgctgaatg gtagcaaaga 360

cgatttcctg atctacgatc gttgtggtcg tctggtatac catctgggtc tgccgttcag 420

ctttctgact ttcccatacg tggaagaagc catcaagatt gcgtactgtg agaagaaatg 480

cggtaactgc tctctgacta ctctgaaaga cgaagacttc tgcaaacgtg taagcctggc 540

tactgttgac aagaccgttg aaactccgtc tccacattac caccatgagc accatcacaa 600

tcatggtcat cagcatctgg gtagctctga actgtctgag aatcagcaac caggtgcacc 660

taatgctccg actcatccag ctcctccagg tctgcatcac catcacaagc acaaaggtca 720

gcatcgtcaa ggtcatccag agaatcgtga tatgcctgca agcgaagatc tgcaagacct 780

gcagaagaaa ctgtgtcgta agcgttgcat caatcaactg ctgtgcaaac tgccgactga 840

tagcgaactg gctccacgta gctgttgctg tcactgtcgt catctgatct tcgagaagac 900

tggttctgca atcacctgtc agtgcaaaga gaacctgcca tctctgtgta gctgccaagg 960

tctgcgtgca gaagagaaca tcactgaatc ctgtcagtgt cgtctgcctc cagctgcatg 1020

ccagatctct cagcaactga ttccgactga agccagcgca tcttgtcgtt gcaagaatca 1080

ggctaagaaa tgcgaatgtc cgtctaacta actcgagg 1118

<210> 2

<211> 367

<212> PRT

<213>artificial sequence

<400> 2

Gly Gly Gly Gly Thr Glu Ser Gln Asp Gln Ser Ser Leu Cys Lys Gln

1 5 10 15

Pro Pro Ala Trp Ser Ile Arg Asp Gln Asp Pro Met Leu Asn Ser Asn

20 25 30

Gly Ser Val Thr Val Val Ala Leu Leu Gln Ala Ser Cys Tyr Leu Cys

35 40 45

Ile Leu Gln Ala Ser Lys Leu Glu Asp Leu Arg Val Lys Leu Lys Lys

50 55 60

Glu Gly Tyr Ser Asn Ile Ser Tyr Ile Val Val Asn His Gln Gly Ile

65 70 75 80

Ser Ser Arg Leu Lys Tyr Thr His Leu Lys Asn Lys Val Ser Glu His

85 90 95

Ile Pro Val Tyr Gln Gln Glu Glu Asn Gln Thr Asp Val Trp Thr Leu

100 105 110

Leu Asn Gly Ser Lys Asp Asp Phe Leu Ile Tyr Asp Arg Cys Gly Arg

115 120 125

Leu Val Tyr His Leu Gly Leu Pro Phe Ser Phe Leu Thr Phe Pro Tyr

130 135 140

Val Glu Glu Ala Ile Lys Ile Ala Tyr Cys Glu Lys Lys Cys Gly Asn

145 150 155 160

Cys Ser Leu Thr Thr Leu Lys Asp Glu Asp Phe Cys Lys Arg Val Ser

165 170 175

Leu Ala Thr Val Asp Lys Thr Val Glu Thr Pro Ser Pro His Tyr His

180 185 190

His Glu His His His Asn His Gly His Gln His Leu Gly Ser Ser Glu

195 200 205

Leu Ser Glu Asn Gln Gln Pro Gly Ala Pro Asn Ala Pro Thr His Pro

210 215 220

Ala Pro Pro Gly Leu His His His His Lys His Lys Gly Gln His Arg

225 230 235 240

Gln Gly His Pro Glu Asn Arg Asp Met Pro Ala Ser Glu Asp Leu Gln

245 250 255

Asp Leu Gln Lys Lys Leu Cys Arg Lys Arg Cys Ile Asn Gln Leu Leu

260 265 270

Cys Lys Leu Pro Thr Asp Ser Glu Leu Ala Pro Arg Ser Cys Cys Cys

275 280 285

His Cys Arg His Leu Ile Phe Glu Lys Thr Gly Ser Ala Ile Thr Cys

290 295 300

Gln Cys Lys Glu Asn Leu Pro Ser Leu Cys Ser Cys Gln Gly Leu Arg

305 310 315 320

Ala Glu Glu Asn Ile Thr Glu Ser Cys Gln Cys Arg Leu Pro Pro Ala

325 330 335

Ala Cys Gln Ile Ser Gln Gln Leu Ile Pro Thr Glu Ala Ser Ala Ser

340 345 350

Cys Arg Cys Lys Asn Gln Ala Lys Lys Cys Glu Cys Pro Ser Asn

355 360 365

Claims

1. the immune detection product of Selenoprotein P, including connection gasket interconnected, absorption pad and with the detection line being separated from each other With the coated film of nature controlling line, the coated film is between the connection gasket and the absorption pad, it is characterised in that: the connection Load has colloid gold label SePm antibody on pad；The detection line is coated with SePm antibody, and the quality inspection line is coated with as anti- Former SePm albumen；

The SePm albumen is following A1), A2) or A3):

A1) amino acid sequence is the protein of sequence 2；

A2) by the amino acid sequence of sequence 2 in sequence table by one or several amino acid residues substitution and/or missing and/ Or addition and protein with the same function；

The polyclonal antibody is the polyclonal antibody obtained using the SePm protein immune animal.

2. product according to claim 1, it is characterised in that: the product also contains sample pad, and the sample pad is located at The non-coated film side of the connection gasket is simultaneously connected with the connection gasket.

3. product according to claim 1 or 2, it is characterised in that: the package amount of the SePm antibody in the detection line For 2 μ g/cm；

And/or the package amount of the SePm antigen is 0.2 μ g/cm on the quality inspection line.

4. product according to claim 1 to 3, it is characterised in that: the preparation method of the SePm albumen includes: The encoding gene of the SePm albumen is imported in microorganism, recombinant microorganism is obtained, makes described in the recombinant microorganism The encoding gene of SePm albumen is expressed, and the SePm albumen is obtained.

5. product according to claim 4, it is characterised in that: the encoding gene of the SePm albumen be b1), b2), b3) Or b4):

B2) coded sequence is 8-1111 DNA moleculars of sequence 1 in sequence table；

B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% or more identity, and encodes the SePm albumen CDNA molecule or DNA molecular；

6. any product in -5 according to claim 1, it is characterised in that: the preparation of the colloid gold label SePm antibody Method includes: to be reacted colloidal gold with the SePm antibody to obtain the colloid gold label SePm antibody.

7. any product in -6 according to claim 1, it is characterised in that: 1), 2) or 3) animal is:

1) mammal；

2) rabbit；

3) mouse.

8. the encoding gene or the SePm antibody of any SePm albumen, the SePm albumen in claim 1-7.

9. the method for detection or auxiliary detection Selenoprotein P, comprising: sample to be tested is added to any production in claim 2-7 In the sample pad of product, whether it is presented whether red determining sample to be tested contains Selenoprotein P according to the detection line of the product: such as institute The detection line and quality inspection line for stating product take on a red color, and the sample to be tested contains or candidate contains Selenoprotein P；Such as the product Detection line does not take on a red color, and quality inspection line takes on a red color, and the sample to be tested does not contain or candidate is without containing Selenoprotein P；Such as the product Detection line and quality inspection line do not take on a red color, the product failure, it is unknown whether the sample to be tested contains Selenoprotein P.

10. following X1 or X2:

Any SePm albumen, the encoding gene of the SePm albumen, the SePm antibody or institute in X1, claim 1-7 State product it is any in following X1a-X1f in application: