CN103175952A - Novel antigen for detecting autoantibodies related to immunological infertility - Google Patents

Abstract

The invention discloses a novel antigen for detecting autoantibodies related to immunological infertility. The invention belongs to the field of reproductive immunology immune infertility. Diagnosis of a laboratory of immunological infertility mainly aims at an anti-sperm antibody at present; however, sperm antigens are composite antigens; most of sperm antigens are unconcerned with infertility; and sperm-specific lactate dehydrogenase is one of sperm antigens with intimate connection with infertility. The human sperm-specific lactate dehydrogenase recombinant vector is built by a molecular cloning method in the research; the transformed bacteria can efficiently express rhLDHC4 protein after the vector is converted into E.coliBL21 under induction of isopropyl thio-beta-D-galactoside; and an indirect enzyme-linked immuno sorbent assay (ELISA) method for human serum anti-LDH-C4 antibody (IgG type) detection is built by the protein after purification. The experimental results show that serum of a childbearing woman and the serum of infertility with unknown causes are detected by the method; and the difference of positive incidences has significant statistical significance.

Description

A kind of novel antigens for detection of the immunological infertility related auto-antibodies

Technical field

Reproductive immunology comprises that the genital tract mucosal immunity is regulated, fertility immunological regulation, mother-tire immunological regulation and reproductive endocrine-immunological regulation 4 aspects.Reproductive immunology thinks that the mankind's gonad and the hormone of reproduction cell and generation thereof all have antigenicity, can cause immune response, produces autoantibody, affects the links of reproductive process.Multiple autoantibody is arranged in normal human serum, but that it is tired is very low, is not enough to cause destruction, therefore be referred to as " physiological antibody "; But after it tires obviously rising, namely cause autoimmune disease.1900, Meichnikoff found that sperm has the generation of antigenicity and energy inducing specific immune response.At first Wilson in 1954 and Rumke have found AsAb (antisperm antibody, AsAb) in the male sterility patients serum.Marshburn in 1994 etc. research and propose by numerous: the AsAb in serum and genital tract can affect the mankind's fecundity.There is research to point out: the sterile 10%-30% that accounts for overall sterile person that is caused by AsAb.Yet sperm antigen is a kind of compositeness antigen, be not all AsAb all with sterile relevant, only have those AsAbs for the target antigen that plays a crucial role in fertilization process just might affect fertility.On sperm with the irrelevant invalid antigen of fertility, may reduce detection sensitivity and specificity, bring unnecessary trouble for patient's treatment.Therefore, separation and purification sperm-specific target antigen as envelope antigen, is the method for setting up the AsAb that detects accurately the impact fertility.

Background technology

Immune infertility typically refers to one of cause of disease of getting rid of " Unexplained infertiiity " outside organic disease clinically, for the laboratory diagnosis of immune infertility, is mainly to measure for AsAb at present.The technology that adopts comprises traditional antigen-antibody reaction, complement fixation test (CFT), immunofluorescence, immunobead, enzyme linked immunosorbent assay, mixed antiglobulin reaction etc., and the clinical samples of detection comprises serum, cervical mucus, refining etc.Yet human sperm's antigen very complex approximately has kind more than 100, is divided into sperm-specific antigen and sperm heterogenetic antigen by its specificity.The AsAb of their generations of inducing is not quite similar to the influence power of fertility.Sperm-specific lactic dehydrogenase (sperm-specific lactate dehydrogenase, LDH-C4) be present in specifically in birds and mammiferous testis and sperm, but the conversion of catalysis pyruvic acid and lactic acid, for motion and the survival of sperm provides energy, the immune system of jenny and buck all can be identified as it dissident's composition, as autoantigen, alloantigen or allogenic antigen, induce body to produce immune response.With the LDHC4 albumen of purifying, through the LDHC4-DNA of the LDHC4 of chemical modification polypeptide or structure vaccine immunity animal used as test, all the existence of specific antibody can be detected in the serum of animal or genital tract juice, and the fertility-rate of animal obviously reduces, and illustrates that LDHC4 antibody may be a kind of antibody closely relevant with reproduction in AsAb.

Summary of the invention

(1) purpose

Prokaryotic expression and purification recombined human sperm-specific lactic dehydrogenase (rhLDH-C4) antigen, and take this antigen as envelope antigen, set up the indirect ELISA method that the anti-LDH-C4 antibody of human serum (IgG type) detects, for the autoantibody that detects more accurately in the immune infertility patient body is set up a kind of reliable method.And the physical relationship of the discussion anti-LDH-C4 antibody of human serum and sterility.

(2) technology path

Structure and the evaluation of 1 pET-28 (a+)-hLDHC recombinant vector

The amplification of purpose fragment and recovery: according to the people LDH-C mRNA(GI:187065 of GenBank login) full length sequence, adopt OMIGA software, designed, designed is for the primer at people LDH-C complete encoding sequence two ends, and entrusts Shanghai Bo Ya company synthetic.The P1(upstream primer): 5 '-CCGAAGCTTGCACCAT

GTCAACTGTCAAGGAGCAGC-3 ' is with a Hind III restriction enzyme site (line place).The P2(downstream primer) sequence be 5 '-CCGCTCGAGTTAAAATATTAGATCCTTT-

TGAATATTCC-3 ' is with an Xhol I restriction enzyme site (line place).Take people's testis λ TripEx cDNA library as template, the pcr amplification reaction cumulative volume that carries out coded sequence is 50 μ l.System is as follows: 10 * PCR damping fluid, 5 μ l, dNTP mixed liquor 4 μ l, cDNA template 1 μ l, each 1 μ l of upstream and downstream primer, Taq polymerase (5U/ μ l) 0.5 μ l, ddH2O 37.5 μ l.After mixing, 94 ℃ of denaturations 4 minutes and 30 seconds are by following parameter: 94 ℃ of 30sec, 55 ℃ of 30sec, 72 ℃ of 2min, amplification.Totally 30 circulations.After finishing, last circulation continues to extend 6min at 72 ℃.Product is electrophoresis in 2.0% Ago-Gel, cuts to contain the gel piece that band is distinguished in expection the Shanghai DNA gel recovery kit recovery DNA of Hua Shun company.Concrete grammar is as follows: with the gel piece that downcuts, put into the eppendorf pipe, every 100mg gel piece adds 300 μ l S1 damping fluids, puts 50 ℃ of water-bath 10min and makes the peptization solution; Solution is moved into adsorption column, the centrifugal 30s of 13000r/min; Abandon centrifugate, add 500 μ l lavation buffer solution W1, the centrifugal 15sec of 13000r/min.Repeat this step once; After the centrifugal 1min of 13000r/min, adsorption column is put into a new 1.5ml eppendorf pipe, add 30 μ l ultrapure waters, after the standing 1min of room temperature, the centrifugal 2min of 13000r/min, centrifugate in collection tube.

The structure of recombinant vector: the PCR product that reclaims is carried out digestion with restriction enzyme by following system: 10 * enzyme cutting buffering liquid, 5 μ l, PCR reclaims product (the 25 μ l of 71 μ g/ml), Hind III (10U/ μ l) 1 μ l, Xhol I (10U/ μ l) 1 μ l, ddH2O 18 μ l.Prokaryotic expression carrier (pET-28a plasmid) is carried out digestion with restriction enzyme by following system: 10 * enzyme cutting buffering liquid, 5 μ l, pET-28a plasmid (265 μ g/ml) 10 μ l, Hind III (10U/ μ l) 2 μ l, Xhol I (10U/ μ l) 2 μ l, ddH2O 31 μ l.

After above-mentioned reaction system difference mixing, react 3h in 37 ℃ of water baths.Enzyme is cut afterproduct and enzyme and is cut front product and do simultaneously the DNA gel electrophoresis, judges whether complete enzyme is cut.Downcut with blade the gel piece contain expection district band under uviol lamp, therefrom reclaim DNA with Shanghai Hua Shun company DNA gel recovery kit.Concrete grammar is the same.The PCR product of the double digestion after reclaiming is connected by following system with the pET-28a plasmid: 10 * connection damping fluid, 1 μ l, pET-28a plasmid (the 2 μ l of 38 μ g/ml), PCR product (the 3 μ l of 36 μ g/ml), T4 DNA ligase 0.5 μ l, ddH2O 3.5 μ l.The reaction cumulative volume is 10 μ l, and 16 ℃ are spent the night.

Express the Preparation and identification of bacterium and clone bacterium: use the CaCl2 facture, preparation E.coliBL21(DE3) the plysS competent cell is as expressing bacterium, concrete steps are as follows: under (1) aseptic condition from the fresh E.coliBL21(DE3 of 37 ℃ of cultivations) the single colony inoculation of picking does not contain in antibiotic LB fluid nutrient medium in 3ml the plysS flat board, 37 ℃ of shaking tables shake bacterium and spend the night.(2) 0.1mol/L CaCl2 and 1.5ml eppendorf pipe are placed on ice bath in the ice bath box.(3) get the above-mentioned bacterium liquid 30 μ l that spend the night and be inoculated in 3ml and do not contain in antibiotic LB fluid nutrient medium, violent jolting 2h on 37 ℃ of shaking tables.(4) bacterium liquid is moved in the 1.5ml eppendorf pipe of ice precooling, put ice bath 5 min in the ice bath box.(5) under 4 ℃ of conditions, the centrifugal 30s of 13000r/min.(6) abandon supernatant, add the 500 μ l 0.1mol/L CaCl2 resuspended bacterium liquid gently of icing precooling, make it become even suspendible shape, put ice bath 5 min in the ice bath box.(7) under 4 ℃ of conditions, the centrifugal 30s of 13000r/min.(8) abandon supernatant, add the 200 μ l 0.1mol/L CaCl2 resuspended bacterium liquid gently of icing precooling, place 24 h for 4 ℃.(9) take out above-mentioned competent cell, add 10 μ l to connect product, suspendible bacterium liquid, put ice bath 30 min in the ice bath box gently.(10) 42 ℃ of water-bath heat shock 90 s(do not shake the EP pipe therebetween, and the shock time must be accurately), fast the EP pipe is put into ice bath box ice bath 5min, make cell cooling.(11) add the LB fluid nutrient medium 900 μ l of 37 ℃ of pre-temperature, 37 ℃ of shaking table gentlenesses are shaken bacterium 45 min, make cell recovery.(12) the centrifugal 30s of 13000r/min discards 700 μ l supernatants.(13) with pipettor with transformed bacteria suspendible gently, all be added drop-wise on the LB culture plate that contains 100mg/L kanamycins (Kan+) of pre-temperature, evenly be coated with aseptic elbow glass bar.After 37 ℃ of incubators placement products to be transformed were absorbed by nutrient culture media fully, inversion was spent the night.(14) observe the colony growth situation next day.Prepare E. coli JM109 clone bacterium by the above-mentioned method of expressing bacterium for preparing.

Under aseptic condition from the fresh flat board of 37 ℃ of cultivations the single colony inoculation of picking do not contain in antibiotic LB fluid nutrient medium in 3ml, 37 ℃ of shaking tables shake bacterium and spend the night.Use Shanghai Hua Shun company plasmid extraction purification kit extracting recombinant plasmid, concrete grammar is as follows: (1) abandons supernatant with the centrifugal 1min of 3ml bacterium liquid 13000r/min of incubated overnight; (2) add 250 μ l P1 liquid, vibration is to thoroughly suspending; (3) add 250 μ l P2 liquid, gentleness is put upside down eppendorf and is managed 5～8 times with mixing, standing 4 min of room temperature immediately; (4) add 350 μ l P3 liquid, gentleness is put upside down eppendorf and is managed 5～8 times with mixing immediately, and the centrifugal 10min of 13000r/min carefully moves supernatant to adsorption column, and the centrifugal 15sec of 13000r/min abandons centrifugate; (5) add 500 μ l B1 liquid, the centrifugal 15sec of 13000r/min abandons centrifugate; (6) add 500 μ l W1 liquid, the centrifugal 15sec of 13000r/min abandons centrifugate; (7) repeating step (6) once, the centrifugal 1min of 13000r/min is placed on adsorption column on new eppendorf pipe; (8) add 50 μ l T1 liquid, standing 1 min of room temperature, the centrifugal 1min of 13000r/min collects liquid in centrifuge tube; (9) get and carry out respectively in right amount DNA and quantitatively identify (reaction system such as front) with Hind III/Xhol I double digestion, whether contain the purpose segment with observation.Recombinant plasmid entrusts Shanghai Bo Ya Bioisystech Co., Ltd to check order.

The prokaryotic expression of 2 rhLDH-C4 proteantigens and purifying

The expression of recombinant protein antigen: under aseptic condition from the fresh flat board of 37 ℃ of cultivations the expression bacterium colony inoculation of the single conversion of picking do not contain in antibiotic LB fluid nutrient medium in 3ml, 37 ℃ of shaking tables shake bacterium and spend the night.Get the 30 μ l bacterium that spends the night and be inoculated in the LB nutrient culture media that 3ml contains Kan, 37 ℃ of shaken cultivation to A600 be 0.4-0.6, add IPTG to final concentration 0.1mM, another does not add IPTG as inducing front contrast, induces 5h for 30 ℃.Then respectively get 0.5ml bacterium liquid, the 13000 centrifugal 3min of rpm abandon supernatant, add the 80 resuspended precipitations of μ l 1 * PBS, then add 5 * sds gel sample-loading buffer, 20 μ l mixings, and 100 ℃ are boiled 5min.The centrifugal 10min of 5000rpm gets supernatant electrophoresis (12%SDS-PAGE) in the 12%SDS polyacrylamide gel.Under 200V voltage after electrophoresis 55min, carefully take out the gel 2-3h that dyes in coomassie brilliant blue staining liquid, decolour clear to background, become in the Flour-S imaging system mutually and preserve.

The purifying of recombinant protein antigen: by above-mentioned abduction delivering condition, the recombinant bacterium liquid 1000ml that gets after inducing is centrifugal, collects thalline, is resuspended in 10ml binding buffer liquid.Adding Triton X-100 100 μ l(final concentrations is 1%) and lysozyme (10mg/ml) 100 μ l, and add appropriate DNA enzyme-I, in liquid nitrogen with 37 ℃ of (in constant water bath box) conditions under 4 cracking thalline of multigelation.Under 4 ℃ of conditions, the centrifugal collection supernatant of 14000rpm * 10min.After carrying out condition optimizing according to His-fusion protein purification kit (Qiagen company product) the described method of instructions, with the Ni+-NTA affinity chromatography, the rhLDH-C4 albumen of solubility in supernatant is carried out separation and purification., detailed process is as follows: (1) gets gained supernatant 10ml and 1ml 50% Ni+-NTA mixing after cellular lysate.Under 4 ℃ of conditions, after gentle vibration makes its abundant combination repeatedly, dress Ni+ post; (2) open Ni+ post lid, the interior liquid of post is flowed out naturally, collect prick post liquid.(3) wash the Ni+ post 3 times with the 25ml lavation buffer solution, collected the post cleansing solution.(4) cross the post eluted protein with elution buffer, each 1ml collects each eluent, and monitoring eluent lactic acid dehydrogenase activity stops wash-out in good time.In purge process, with the lactic acid dehydrogenase activity in thalline supernatant and prick post liquid, cleansing solution, eluent before OLYMPUS AU2700 automatic biochemistry analyzer monitoring purifying.To not induce thalline, induce rear thalline and prick post liquid, cleansing solution, eluent etc. carry out 12%SDS-PAGE and identify its internal protein composition.

The concentrated of purifying protein reaches quantitatively: with the recombinant protein liquid of above-mentioned purifying gained, pack in a dialysis band, tighten with line at two ends, after the PBS dialysis equilibrium, imbeds in the plate that sucrose is housed.Until liquid concentration when the desirable volume, carry out protein quantification with protein quantification kit (PIERCE company product), concrete grammar is as follows: (1) provides standard items to prepare protein concentration and absorbance typical curve with kit, and input trace dna-protein quantification instrument is standby.(2) the A liquid that kit is provided mixes according to the 50:1 ratio with B liquid, is working fluid.(3) 25 μ l testing samples are added in 200 μ l working fluids 37 ℃ of water-bath 30min.(4) be cooled to room temperature, survey absorbance at wavelength 562nm place, trace dna-protein quantification instrument protein concentration that automatically converts.25 μ l PBS add in 200 μ l working fluids and return to zero as blank.Get a certain amount of concentrated purifying protein after quantitatively and carry out the SDS-PAGE electrophoresis, observe the Zone electophoresis band after coomassie brilliant blue staining, with the purity of this purification Identification albumen.1000ml bacterium liquid can obtain approximately 1.5mg of destination protein altogether.

The foundation of the 3 anti-LDH-C4 antibody of human serum (IgG type) indirect ELISA methods

Enzyme labelled antibody is determining of the suitableeest working concentration: (1) is with 100ng/ml human IgG coated elisa plate (4 ℃ of 18-24h), PBS washing 3 times, each 5min.(2) HRP mark mouse-anti human IgG is done a series of dilutions (1:2500-1:80000) with 1% BSA-PBS dilution, add respectively IgG to be coated with in the hole, every hole 100 μ l, under room temperature, after reaction 2h, PBS washs 3 times, each 5min.(3) add substrate H2O2/TMB, incubate for 37 ℃ and bathe 20min, survey the OD value after adding 2M H2SO4 cessation reaction.(4) according to document, take the OD495 value as enzyme mark mouse-anti human IgG antibody's dilutability (1:20000) of 1.0 o'clock for the suitableeest working concentration.

Antigen is determining of the suitableeest coated concentration: (1) with rhLDH-C4 antigen take the carbonate buffer solution dilution of pH9.6 as a series of concentration such as 0.000,0.125,0.250,0.500,1.000,2.000 and 4.000 μ g/ml, then be added in the micro-hole of ELISA Plate, each coated 6 holes of the antigen of each concentration, every hole 100 μ l.(2) ELISA Plate is put 4 ℃ coated spend the night (18-24h).(3) take out plate, with PBS washing 3 times, each 3min, button is done.(4) every hole adds the confining liquid of 200 μ l, ELISA Plate is put in wet box, in 37 ℃ of sealing 2h.(5) take out plate, with PBS washing 3 times, each 3min, button is done.(6) each coated concentration adds respectively positive control serum (PC), negative control sera (NC) and the dilution (blank) of 1:100 dilution, each 2 holes, every hole 100 μ l.(7) ELISA Plate is put in wet box, in 37 ℃ of reaction 1h.With 0.05%Tween-20-PBS washing 5 times, each 3min, button is done.(8) every hole adds the enzyme labelled antibody of 1:20000 dilution, every hole 100 μ l.Put in wet box, in 37 ℃ of reaction 1h.(9) the PBS washing is 5 times, each 3min, and button is done.Every hole adds the substrate solution of 100 μ l, 37 ℃ of effect 20min.(10) with 2M H2SO4 cessation reaction, every hole 50 μ l.(11) read each hole OD value under the wavelength of 495nm, with OD positive control/OD negative control ratio the maximum for the suitableeest antigen coated concentration (1.000 μ g/ml).

Determining of critical value (cut-off): with the suitableeest antigen coated concentration and the enzyme labelled antibody working concentration of determining, measure 25 parts of virgins (less than 10 years old) serum specimen OD value, calculate its mean () and standard deviation (SD).Mean value with virgin's serum specimen OD value adds 3 standard deviations as the judgement critical value (cut-off) of positive serum sample, i.e. cut-off=+3SD; OD value 〉=cut-off is judged to be the positive, and OD value＜cut-off is judged to be feminine gender.

Repeatability detects: the ELISA method of getting foundation detects positive and negative each portion of serum specimen, measures simultaneously respectively 4 holes, calculates its variation within batch coefficient; Different time points is successively measured with a negative serum sample with a positive serum sample for 4 times, calculates its differences between batches; When batch between and batch in difference all less than 10% the time, illustrate that the ELISA method of setting up has good stability.

(3) effect

With 100ng/ml human IgG coated elisa plate, determine that with mouse-anti human IgG (HRP) the enzyme labelled antibody reaction of a series of dilutions is rear 1:20000 is the suitableeest working concentration of enzyme labelled antibody, 1ug/ml is the suitableeest coated concentration of rhLDH-C4 antigen, judge critical value (CUT-OFF) be defined as 0.110, in the repeatability detection display is criticized, differences between batches illustrate that all less than 10% the indirect ELISA method repeatability of setting up is good.Method and the ASA immune colloid gold detection kit of using this experiment foundation detect 47 parts of serum specimens simultaneously, and ASA immune colloid gold detection kit positive rate is 21.28%.And the indirect ELISA method Positive rate of anti-LDH-C4 antibody test is 40.43%, and SPSS 12.0 statistical softwares calculate, and chi-square value (χ 2) is 4.27, P＜0.05, illustrates that there is notable difference in two kinds of detection method testing results.Trace it to its cause and may be: that the immune colloid gold detection kit detects be compound ASA, its envelope antigen used is a kind of complex antigen (being the solubility Sperm Antigen), its antigenic component is complicated, most of antigen and sterile irrelevant wherein, even a part is lost due to the defective of manufacture craft with sterile closely-related antigenic component or the content reduction, therefore its testing result is often unreliable, lack clinical reference value.And the ELISA method that this experiment is set up is as envelope antigen with single sperm-specific rhLDH-C4, only detect for Serum LDH-C4 antibody, it has avoided being mixed with the interference that other non-specific protein ingredient causes result in the envelope antigen, has improved widely the susceptibility and the specificity that detect.

Four embodiments

The indirect ELISA method of setting up with this research detects 74 parts and does not educate person's serum specimen and 177 parts of Unexplained infertiiity person serum specimens.Result shows: sterile person's serum specimen positive rate is 30.51%, and not educating person's serum specimen positive rate is only 9.46%.Application SPSS 12.0 statistical softwares calculate, chi-square value (χ 2) is 12.57, P＜0.005, illustrate that both have remarkable statistical significance at the positive rate difference, do not educate person's serum specimen occur the positive may be due to: the fertility after due to a variety of causes, educated in Mr. and Mrs' body immune infertility occured, and when sampling will its as educating.

Claims (3)

1. this research people's sperm-specific lactic dehydrogenase (sperm-specific lactate dehydrogenases that will clone from people's testis λ TripEx cDNA library, LDH-C4) be connected with efficient prokaryotic expression carrier pET-28a (+), built pET-28 (a+)-hLDHC recombinant plasmid, this Plasmid Transformation is entered Escherichia coli Ecol.BL21(DE3) express in the plysS lysogenic bacteria, induce lower rhLDH-C4 albumen that can high efficient expression solubility at 0.1mM IPTG, after concentrating through Ni+-NTA affinity chromatography purifying and with the dialysis band, concentration can reach 1.0mg/ml, 1000ml bacterium liquid can obtain approximately 1.5mg of destination protein altogether, set up as envelope antigen the indirect ELISA method that the anti-LDH-C4 antibody of human serum (IgG type) detects with this purifying protein, for the autoantibody (LDH-C4 antibody) that detects accurately in the infertile patients body provides a kind of reliable clinical reference index, therefore require as follows:

(1) preservation and the use of pET-28 (a+)-hLDHC RT-PCR carrier.

2. (2) abduction delivering of rhLDH-C4 recombinant protein and purification technique.

3. (3) the rhLDH-C4 purifying protein clinically application and as the new kit of development of raw materials.