CN109021091B - Antigen for detecting sperm antibody, expression vector thereof, kit and detection method - Google Patents
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Abstract
The invention relates to the technical field of reproductive medicine and immune application, in particular to an antigen for detecting sperm antibody, an expression vector thereof, a kit and a detection method. The antigen for detecting sperm antibody is polypeptide encoded by a gene segment of a PB functional region of a recombined IZUMO gene, and the sequence of the polypeptide has a sequence shown by SEQ ID NO. 1. The antigen for detecting the sperm antibody is a key protein of sperm acrosome reaction, determines whether fertilization is successful or not, and the antibody aiming at the antigen is one of important reasons for causing immune infertility. Therefore, the invention is expected to be used for screening and diagnosing the anti-sperm antibody of the infertility patient.
Description
Technical Field
The invention relates to the technical field of reproductive medicine and immune application, in particular to an antigen for detecting sperm antibody, an expression vector thereof, a kit and a detection method.
Background
Infertility (infertility) means a state in which a couple lives together for at least 1 year after marrying, has normal sexual life, and cannot live without any contraceptive measures. The World Health Organization (WHO) has adopted the standard diagnostic method for infertility in 33 centers of 25 countries at the end of the 80 th 20 th century, and the survey data shows that about 6000 to 8000 thousand couples worldwide are in sterile and infertile state, and due to the influence of factors such as environment, hormone and related diseases, the number of the infertile couples is increasing, and about 8 to 12 percent of couples worldwide have experience of infertility. Relevant researches show that the fertility of breeding age couples in China shows a descending trend, and infertility caused by low fertility tends to rise. For a pair of healthy couples, the conception rate of the couple in one month under the natural state is 20-25%.
The infertility is mainly classified into primary infertility and secondary infertility. Primary infertility is never acquired, while secondary infertility is never acquired after a previous pregnancy. With the change of social environment, the development of economy and the increasing pressure, more couples face the problem of infertility, about 10-15% of couples in the reproductive age are affected, and the reasons for causing the infertility can be divided into male infertility and female infertility. The main causes of infertility are in turn: ovulation disorders, semen abnormalities, fallopian tube abnormalities, infertility of unknown origin, endometriosis and others such as immunological infertility. Female infertility is mainly caused by ovulation failure, fallopian tube factors and endometrial receptivity abnormality, while male infertility is mainly caused by spermatogenesis abnormality.
The examination of infertility includes correct collection of medical history, understanding of occupation and work, past medical history, marital and sexual life conditions, past examination and treatment conditions, family history, etc. of patients. Physical examination includes examination of the whole body and reproductive organs. Male laboratory examinations including routine semen analysis, prostate fluid examination; endocrine examination includes examining the function of the hypothalamus-pituitary-testicular axis by gonadotropin releasing hormone or the stimulation test of the clavifenesin; doppler ultrasound examination helps to confirm varicocele; the X-ray examination can determine the infarct position of the insemination pipeline; and immunological tests. The female laboratory examination comprises the steps of adopting B-type ultrasonic to examine the development condition and the morphological position of the uterus and the accessories, and whether pathological changes exist, such as endometriosis, ovarian tumor, fallopian tube tumor, uterine fibroid and the like; checking the patency of the oviduct by adopting a dynamic iodized oil angiography of the uterine oviduct; performing an examination of an anti-sperm antibody in blood and an anti-sperm antibody against cervical mucus in a patient suspected of immune infertility using an immunoassay; measuring endocrine related hormones such as Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Prolactin (PRL), estradiol (E2) and the like by fasting blood sampling at the preovulatory and preovulatory stages, or detecting blood progesterone (P) at the middle section of the basal body temperature high-temperature phase, adopting laparoscopy for examination, and treating under direct vision while carrying out laparoscopy for examination.
The immunological examination is mainly used for examining immune infertility, and the immune infertility refers to infertility caused by autoimmunity or alloimmunity of reproductive system antigens. According to statistics, the infertility caused by immunological factors accounts for about 10-20% of all causes causing the infertility. The laboratory tests generally include antiphospholipid antibodies (APA), antinuclear antibodies (ANA), anti-DNA antibodies, antisperm antibodies (ASAb), anti-endometrial antibodies (AEMAb), anti-chorionic gonadotropin antibodies (AhCGab), anti-ovarian antibodies (AOVAb), and the like. It has been found that 79% of infertile women with unknown cause have antisperm antibodies in their body; among patients with infertility, 16% of male patients and 29% of female patients contain antisperm antibodies, and the female patients have higher antibody incidence rate than the male patients and have more strong positive patients.
Sperm has the possibility of becoming self-antigen and alloantigen, sperm antigen can generate antisperm antibody (ASA) in human body, and sperm polypeptide antigen has a plurality of antigens, such as P10G, YLP12, SP17, IR9, LDH-C4, EPPIN, IZUMO protein and the like. Wherein the IZUMO protein is specifically expressed in the sperm cell membrane, the sperm surface can be combined with the anti-IZUMO antibody only after acrosome reaction, and the IZUMO protein can not be detected on the sperm surface without acrosome reaction. This is because when sperm is capacitated, after the ampulla of the oviduct meets the ovum, a series of changes occur in the acrosome, so that the IZUMO protein is exposed from the spermatic membrane and combined with juno on the surface of the ovum, and finally the sperm-ovum fusion is completed to form a fertilized ovum. Therefore, the IZUMO protein plays an important role in the fertilization process. If anti-IZUMO antibodies are produced in vivo, sperm and egg cells will not be able to bind and a fertilized egg will not be formed.
The IZUMO protein is named in japanese bible and means "marriage". The IZUMO protein contains 377 amino acids and is divided into a large fragment extracellular region, a transmembrane region and a small fragment intracellular region. The IZUMO protein is found to be highly conserved in mammals in mice, for example, the human and mouse IZUMO proteins have 57% amino acid sequence identity. The IZUMO protein can be divided into three functional regions of PA, PB and PC, wherein Ig-like domains in the PB region (amino acids 167-253) have stronger immunogenicity and are main regions for inducing an organism to generate antibodies. B cell epitope of mouse IZUMO (YSFYRV)196-201) Is exactly the same as human. The study finds that the mice inoculated with the IZUMO vaccine can play a contraceptive role. That is, if the body produces the antibody, infertility is caused, and therefore, it is presumed that the production of the IZUMO antibody may be a significant cause of infertility.
The test method for detecting the anti-sperm antibody has more than 10 kinds, such as a tray agglutination test, a micro sperm immobilization test, a solid phase immunoadsorption test, an indirect immunofluorescence test, a gelatin agglutination test, an enzyme linked immunosorbent assay, an attrition cell technology method and the like, and the sensitivity, the specificity and the repeatability of different methods are different. The ideal method for detecting anti-sperm antibodies would be: firstly, determining the type of immunoglobulin; ② the antibody can be quantified; thirdly, the combination part of the antibody on the sperm can be judged; and fourthly, the method has good sensitivity, specificity and repeatability. The ELISA technology has the advantages of short time consumption, low cost, mature technology and the like, can be used for detecting clinical specimens in large scale and is widely applied at home and abroad at present. The detection rate of the anti-sperm antibody of the infertility patient is 10-30%, and about 21.5% of the anti-sperm antibody positive rate can be detected by adopting an imported ELISA kit. However, the domestic common ELISA kit has high false positive rate or low positive rate, and the sensitivity and accuracy of the domestic kit are not reliable, so that the requirement of the clinical sterility detection cannot be met, and therefore, a new kit for detecting the anti-sperm antibody with low cost and reliable detection result is urgently needed to meet the clinical requirement.
Disclosure of Invention
The present invention has an object to provide an antigen for detecting sperm antibodies, which addresses the deficiencies of the prior art.
The second object of the present invention is to provide an expression vector for detecting an antigen of a sperm antibody, which is directed against the deficiencies of the prior art.
The present invention also aims to provide a method for constructing an expression vector for detecting an antigen of a sperm antibody, which is directed against the disadvantages of the prior art.
The fourth purpose of the present invention is to provide an ELISA kit for antisperm antibody detection against the deficiencies of the prior art.
The fifth purpose of the present invention is to provide a detection method of ELISA kit for antisperm antibody detection, which is aimed at the defects of the prior art.
In order to achieve one of the purposes, the invention adopts the following technical scheme:
provides an antigen for detecting sperm antibody, wherein the antigen for detecting sperm antibody is polypeptide coded by a PB functional region gene segment of a recombined IZUMO gene, the amino acid sequence of the antigen has a sequence shown by SEQ ID NO. 1, and the gene sequence of the antigen has a sequence shown by SEQ ID NO. 2.
In order to achieve the second purpose, the invention adopts the following technical scheme:
an expression vector for detecting the antigen of sperm antibody is provided, which is named as pET30a-IZUMO-His, and the construction diagram of the expression vector is shown in figure 1.
In order to achieve the third purpose, the invention adopts the following technical scheme:
provides a construction method of an expression vector for detecting an antigen of a sperm antibody, which comprises the following steps:
step one, synthesizing a gene fragment of a PB functional region of a recombinant IZUMO gene: obtaining a PB gene sequence by adopting a gene synthesis mode, and inserting a His label at the 3' end to obtain a gene segment with a sequence shown as SEQ ID NO. 1;
step two, PB gene fragment amplification: and (4) carrying out PCR amplification by taking the gene fragment obtained in the step one as a template to obtain the expression vector of the antigen for detecting the sperm antibody.
In the above technical solution, in the second step, the PCR amplification conditions are: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 40s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 45s, 30 cycles, and extension at 72 ℃ for 10 min.
In order to achieve the fourth purpose, the invention adopts the following technical scheme:
provides an ELISA kit for detecting an anti-sperm antibody, which comprises the following components: the kit comprises the detachable enzyme label plate coated by the antigen for detecting the sperm antibody, a positive standard substance, a negative control substance, an anti-antibody marked by horseradish peroxidase, a developing solution and a stop solution.
The detachable enzyme label plate is a 96-hole corning detachable enzyme label plate; the positive standard substance is a rabbit anti-human IZUMO recombinant sperm antigen PB functional region polypeptide antibody, and the negative control substance is virgin serum.
In order to achieve the fifth purpose, the invention adopts the following technical scheme:
provides a detection method of an ELISA kit for detecting an anti-sperm antibody, which comprises the following steps:
step one, coating: diluting the target protein by using a coating buffer solution, coating the diluted protein-containing coating solution on a 96-well enzyme label plate by using 100 mu L/well, incubating for 12h at 4 ℃, washing for 5 times by using a washing solution, slightly shaking by holding the plate by hands each time, paying attention to not spill to pollute adjacent wells, washing, throwing off the washing solution, and patting to dry;
step two, sealing: adding 250 mu L of confining liquid into each hole, incubating for 2h at 37 ℃ or incubating for 12 h-16 h at 4 ℃, washing the plate and drying for 5 times;
step three, adding a sample: adding samples, incubating for 1h at 37 ℃ with 100 mu L of each well, washing the plate for 5 times, and drying;
step four, adding a horseradish peroxidase-labeled secondary antibody: adding a mouse anti-human IgG monoclonal antibody with a certain dilution into each hole, incubating for 1h at 37 ℃ with 100 mu L of each hole, washing the plate and drying for 5 times;
step five, color development: adding a one-step color-developing agent TMB, incubating for 10-25 min at 37 ℃ in a dark place, wherein each hole is 50 mu L;
step six, terminating the reaction: adding 50 mu L of ELISA stop solution into each reaction hole, and finishing color comparison within 5 minutes;
step seven, color comparison: the OD value of each well at the detection wavelength of 450 nm.
In the above technical solution, in the first step, in the diluted coating solution containing protein, the protein concentration of the coating solution is 5 ng/mL;
the coating buffer solution is sodium carbonate buffer solution or PBS;
the sodium carbonate buffer solution is prepared by the following steps: na (Na)2CO3 1.59g，NaHCO32.93g, adding double distilled water to a constant volume of 1000mL, adjusting the pH value to 9.6, and storing in a refrigerator at 4 ℃;
the PBS was 0.01M phosphate buffer.
In the above technical solution, in the second step, the blocking solution is one of 3% mass concentration PBS, 5% mass concentration PBS, 3% mass concentration PBST, or 5% mass concentration PBST.
In the above technical scheme, in the fourth step, the dilution of the mouse anti-human IgG monoclonal antibody is 1: 5000.
Compared with the prior art, the invention has the beneficial effects that:
(1) the antigen for detecting the sperm antibody is a key protein of sperm acrosome reaction and determines whether fertilization is successful or not, and the antibody aiming at the antigen is one of important reasons for causing immune infertility. Therefore, the invention is expected to be used for screening and diagnosing the anti-sperm antibody of the infertility patient.
(2) The ELISA kit for detecting the anti-sperm antibody has the advantages of low price and reliable detection result.
(3) The invention provides a detection method of an ELISA kit for detecting an anti-sperm antibody, which is an ELISA method established by taking recombinant human sperm IZUMO protein as an antigen, and can be used for diagnosing clinical immune infertility by detecting clinical samples.
Drawings
FIG. 1 is a diagram showing the construction of an expression vector for detecting an antigen of a sperm antibody according to the present invention.
FIG. 2 is a diagram showing the PCR results of a PB gene fragment for detecting an antigen of a sperm antibody according to the present invention. Wherein M is DNA marker; PB is PB gene PCR product.
FIG. 3 is a diagram showing the results of double digestion of the recombinant plasmid pET30a-IZUMO-His according to the present invention. Wherein, M represents a DNA Marker; 1 represents the result of electrophoresis of pET30a-IZUMO-His recombinant plasmid; 2 represents the result of double-restriction electrophoresis of pET30a-IZUMO-His recombinant plasmid.
FIG. 4 is a graph showing the degree of match of the nucleotide sequence of the positive clone pET30a-IZUMO-His plasmid.
FIG. 5 is an SDS-PAGE analysis of the expression of the recombinant PB protein of the invention in BL21(DE 3). Wherein lane M1 represents protein marker; lane PC1 represents BSA (1. mu.g); lane PC2 represents BSA (2. mu.g); lane NC represents whole cell lysate; lane 1 represents a whole cell lysate at 15 ℃ for 16 hours; lane 2 represents a whole cell lysate at 37 ℃ for 4 hours; lane NC1 represents cell lysate supernatant; lane NC2 represents cell lysate pellet; lane 3 represents the supernatant of cell lysate at 15 ℃ for 16 hours; lane 4 represents cell lysate pellet at 15 ℃ for 16 hours; lane 5 represents the supernatant of the cell lysate at 37 ℃ for 4 hours; lane 6 represents cell lysate pellets at 37 ℃ for 4 hours.
FIG. 6 shows Western blot analysis of expression of the recombinant PB protein of the invention in BL21(DE 3). Wherein lane M1 represents protein marker; lane 1 represents cell lysate pellets at 37 ℃ for 4 hours; lane 2 represents the supernatant of the cell lysate at 37 ℃ for 4 hours; lane 3 represents cell lysate pellet at 15 ℃ for 16 hours; lane 4 represents cell lysate supernatant at 15 ℃ for 16 hours; lane 2 represents a whole cell lysate at 37 ℃ for 4 hours; lane 6 represents a whole cell lysate at 15 ℃ for 16 hours.
FIG. 7 is an SDS-PAGE analysis of recombinant protein purification of the present invention. Wherein lane 1 is the positive control BSA, lane 2 represents the purified PB protein, and M represents the protein marker.
FIG. 8 is a Western blot analysis chart of recombinant protein purification of the present invention. Wherein PB represents purified PB protein and M represents protein marker after the purified PB protein is subjected to color development by the action of an anti-His antibody.
FIG. 9 is a drawing of the purified IZUMO recombinant protein by Western blot detection. Wherein, 1 represents that the purified IZUMO protein is combined with an anti-IZUMO antibody for color development; 2 represents the liquid after 4 th nickel column purification, which contains almost no IZUMO recombinant protein.
FIG. 10 is a schematic diagram of an indirect ELISA assay of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1.
An antigen for detecting sperm antibody is a polypeptide coded by a gene segment of a PB functional region of a recombined IZUMO gene, the amino acid sequence of the antigen has a sequence shown by SEQ ID NO. 1, and the gene sequence of the antigen has a sequence shown by SEQ ID NO. 2.
Example 2.
An expression vector for detecting sperm antibody antigen of example 1, which is named as pET30a-IZUMO-His, and whose construction diagram is shown in figure 1.
Example 3.
The method for constructing an expression vector for detecting an antigen of a sperm antibody of example 2, comprising the steps of:
step one, synthesizing a gene fragment of a PB functional region of a recombinant IZUMO gene: obtaining a PB gene sequence by adopting a gene synthesis mode, and inserting a His label at the 3' end to obtain a gene segment with a sequence shown as SEQ ID NO. 1;
step two, PB gene fragment amplification: and (4) carrying out PCR amplification by taking the gene fragment obtained in the step one as a template to obtain the expression vector of the antigen for detecting the sperm antibody.
In the second step, the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 40s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 45s, 30 cycles, and extension at 72 ℃ for 10 min. The reaction system is shown in table 1.
TABLE 1 recombinant IZUMO Gene amplification PCR reaction System (25. mu.L)
Example 4.
An ELISA kit for detecting an anti-sperm antibody comprises the following components: a detachable elisa plate coated with the antigen for detecting sperm antibody of example 1, a positive standard, a negative control, an anti-antibody labeled with horseradish peroxidase, a developing solution and a stop solution.
Wherein, the detachable ELISA plate is a 96-hole corning detachable ELISA plate; the positive standard substance is a rabbit anti-human IZUMO recombinant sperm antigen PB functional region polypeptide antibody, and the negative control substance is virgin serum.
Example 5.
The detection method of the ELISA kit for detecting the anti-sperm antibody in the embodiment 4 comprises the following steps:
step one, coating: diluting the target protein by using a coating buffer solution, coating the diluted protein-containing coating solution on a 96-well enzyme label plate by using 100 mu L/well, incubating for 12h at 4 ℃, washing for 5 times by using a washing solution, slightly shaking by holding the plate by hands each time, paying attention to not spill to pollute adjacent wells, washing, throwing off the washing solution, and patting to dry;
step two, sealing: adding 250 mu L of confining liquid into each hole, incubating for 2h at 37 ℃ or incubating for 12 h-16 h at 4 ℃, washing the plate and drying for 5 times;
step three, adding a sample: adding samples, incubating for 1h at 37 ℃ with 100 mu L of each well, washing the plate for 5 times, and drying;
step four, adding a horseradish peroxidase-labeled secondary antibody: adding a mouse anti-human IgG monoclonal antibody with a certain dilution into each hole, incubating for 1h at 37 ℃ with 100 mu L of each hole, washing the plate and drying for 5 times;
step five, color development: adding a one-step color-developing agent TMB, incubating for 10-25 min at 37 ℃ in a dark place, wherein each hole is 50 mu L;
step six, terminating the reaction: adding 50 mu L of ELISA stop solution into each reaction hole, and finishing color comparison within 5 minutes;
step seven, color comparison: the OD value of each well at the detection wavelength of 450 nm.
In the first step, the protein concentration of the coating liquid in the diluted coating liquid containing the protein is 5 ng/mL;
wherein the coating buffer solution is sodium carbonate buffer solution or PBS;
wherein, the configuration of the sodium carbonate buffer solution is as follows: na (Na)2CO3 1.59g，NaHCO32.93g, adding double distilled water to a constant volume of 1000mL, adjusting the pH value to 9.6, and storing in a refrigerator at 4 ℃;
wherein the PBS is 0.01M phosphate buffer solution.
In the second step, the blocking solution is one of 3% mass concentration PBS, 5% mass concentration PBS, 3% mass concentration PBST or 5% mass concentration PBST.
Wherein, in the fourth step, the dilution of the mouse anti-human IgG monoclonal antibody is 1: 5000.
Experiment:
experiment one:
1.1 the PCR product obtained in example 3 was identified as follows:
and identifying the PCR product by agar gel electrophoresis: the product was pipetted 5. mu.L of sample, mixed with 1. mu.L of nucleic acid type II dye and 1. mu.L of sample buffer; DNA Maker 2.5. mu.L mixed with 1. mu.L nucleic acid type II dye, electrophoresis voltage 110V, time 40min, observing the position of the strip under a gel imager. The electrophoresis results are shown in FIG. 2.
1.2 recovery of PCR products as follows:
the gel electrophoresis verifies that the size of the result is consistent with that of the target gene fragment, the sample loading amount of the product is enlarged, the gel electrophoresis is carried out, and a TaKaRa gel recovery kit is used, and the steps are as follows: cutting an agarose gel containing a target fragment, cutting the agarose gel, placing the cut agarose gel in a 1.5mL EP tube, weighing the agarose gel by an electronic scale, and converting the volume of the gel block (1mg to 1. mu.L); adding GM Buffer with the volume of 3-5 times of the glue, fully and uniformly mixing, and melting glue blocks in water bath at 37 ℃; pouring the melted glue into a centrifugal tube arranged in a collecting tube, centrifuging for 30s at 10000g, and discarding filtrate; adding 700 mu L of WB Buffer, standing at room temperature for 3-5 minutes, centrifuging at 10000g for 30s, and discarding the filtrate; fifthly, centrifuging once by 10000g of an empty tube, putting a centrifugal column into a new EP tube, adding 30 mu LElution Buffer, standing for 1 minute at room temperature, centrifuging for 2 minutes by 10000g, and obtaining filtrate in a centrifuge tube, namely recovering the target DNA product, and keeping at-20 ℃ for later use.
1.3 PCR product and pET30a plasmid were double digested:
the vector pET30a with the molecular size of 5422bp and multiple cloning sites and containing kanamycin resistance is selected for the experiment. The PCR product of the target gene and the plasmid pET30a were added to two EP tubes, respectively, and restriction enzymes NdeI and HindIII were added thereto, respectively, in a water bath at 37 ℃ for 1.5 hours, and the reaction system is shown in Table 2. And (5) carrying out gel electrophoresis verification after enzyme digestion, and carrying out gel recovery. Wherein, the double digestion result of pET30a-IZUMO-His recombinant plasmid is shown in FIG. 3.
TABLE 2 PCR double digestion reaction System for target genes
1.4 cloning of the target Gene into pET30a vector:
the PCR fragment of the target gene after enzyme digestion and the large fragment of the vector pET30a after double enzyme digestion are added into a 1.5mL EP tube, T4DNA ligase is added, the mixture is gently mixed and then connected at 16 ℃ overnight, and the reaction system is shown in the following table 3.
TABLE 3 DNA ligation System (10. mu.L)
Experiment II, expression of IZUMO protein PB, and identification of the protein PB:
2.1 little expression and solubility analysis of recombinant IZUMO protein:
performing amplification culture on the strain solution with the correct sequencing, sucking 300 μ L of the strain solution, inoculating into 30mL of liquid LB medium, adding 30 μ L of kanamycin, performing shake culture at 37 deg.C and 200rpm overnight, and culturing the strain solution to OD600When the concentration reached about 0.6, 2mL of the bacterial solution was aspirated, and the mixture was incubated at 4 ℃ and 1mL of the suspension was preserved. As a blank control after IPTG induction, 5mL of bacterial solution is not added with an inducer, after centrifugation, lysate is resuspended, and ultrasonic disruption (ice bath 400W, work for 3s, stop for 5s, and disruption for 25min) is carried out to obtain uninduced whole cell lysate, cell lysate supernatant and cell lysate sediment. Adding IPTG (isopropyl thiogalactoside) with the final concentration of 1mmol/L into the rest bacterial liquid, dividing the bacterial liquid into 2 tubes, inducing each tube by 10mL at 15 ℃ for 16h and 37 ℃ for 4h, centrifuging the induced bacterial liquid for 20min at 10000g, and collecting thalli. And (3) resuspending the thallus precipitate by using a lysate, adding 1mL of the lysate into every 15mL of bacteria liquid, and ultrasonically crushing the resuspended thallus to obtain a whole cell lysate, a cell supernatant lysate and a cell precipitate lysate of 37 ℃ and 16 ℃ respectively. The sample loading of each sample was 10. mu.L, and 10. mu.L of 5 × loading buffer was added simultaneously and mixed well, boiled for 10min, electrophoresed by SDS-PAGE at 4 ℃ with 20. mu.L loading per well, 80V for 30min, and the voltage was changed to 120V when the proteins were pressed into a line, and the time was 1 h. And adding Coomassie brilliant blue R250 into the gel after electrophoresis to dye for 2h, taking out the gel, putting the gel into a decolorizing solution to decolorize overnight, replacing the decolorizing solution for several times, observing an electrophoresis result in a gel imager after decolorization, and observing whether the target protein is mainly in supernatant or precipitate of the lysate so as to determine whether the protein is soluble expression or inclusion body expression.
As shown in FIG. 5, SDS-PAGE results showed that after IPTG induction, the engineered bacteria had an exogenous high expression band at the relative molecular mass of 10kD, which is consistent with the expected molecular weight of recombinant protein containing 6 XHis. On the other hand, the E coli BL21(DE3) transformed with the empty vector pET30a plasmid showed no exogenous protein band at a relative molecular mass of 10kD under IPTG induction. And the Escherichia coli can efficiently express the IZUMO recombinant protein through the induction of IPTG for 4 hours at 37 ℃. Analysis of the cell lysate pellet and supernatant showed that IZUMO was mainly present in the cell lysate pellet and was less soluble in the cell lysate pellet.
2.2 Western blotting detection of target protein:
performing membrane transfer on protein gel subjected to SDS-PAGE electrophoresis, wherein a primary antibody is an anti-His-tag mouse monoclonal antibody, and the dilution concentration is 1: 1000; the secondary antibody is rabbit anti-mouse IgG marked by HRP, the dilution concentration is 1:1000, and the secondary antibody is finally exposed and photographed in a gel imager to analyze immunoreaction strips.
The method comprises the following specific steps: firstly, cutting off concentrated gel of protein gel after electrophoresis, and soaking the gel in membrane-transferring buffer solution for half an hour; preparing a PVDF transfer membrane with the same size as the gel, soaking the PVDF transfer membrane in anhydrous methanol for 5min, and then slowly putting the transfer membrane and the filter paper into a transfer membrane buffer solution for soaking and balancing for 15 min; ③ according to the sequence from bottom to top: 3 layers of filter paper, PVDF membrane, glue and 3 layers of filter paper are placed in an electrophoresis tank, an ice bag is placed in the tank, membrane-transferring buffer solution is poured into the tank, the tank works in a refrigerator at 4 ℃, and the membrane is transferred for 90min at 300 mA; fourthly, after the membrane transfer is finished, taking out the PVDF membrane, and washing 3 times by TBS; fifthly, putting the transfer membrane with the protein side facing upwards into 5% skimmed milk powder, and sealing for 1h on a shaking table at 37 ℃ and 80 rpm; sixthly, preparing the primary antibody sealing solution at a dilution ratio of 1:1000, and reacting l h with the PVDF membrane at room temperature; seventhly, washing the PVDF film by TBST for 3 times and 5 min/time; preparing a secondary antibody with TBST solution in a ratio of 1:1000, transferring the membrane into the secondary antibody solution, and reacting for 1h at room temperature; ninthly, washing the PVDF film 3 times by TBST, 6min each time, and exposing: immunoreactive bands were detected by exposure to ECL in a gel imaging system.
The result is shown in figure 6, Western blot analysis is carried out on the engineering bacteria lysate sediment and the supernatant induced by IPTG, and detection by an anti-His antibody (Kinsry, Cat. No. A00186) shows that a specific band exists at the position of 10kD relative to the molecular mass.
2.3 recombinant protein mass expression and inclusion body washing:
and (3) carrying out amplification culture on the bacterial liquid transformed by the expression vector: 60 mu L of kanamycin, 600 mu L of strain and 600mL of LB liquid medium, and shaking-culturing at 37 ℃ for 12 h. Protein expression conditions and protein expression patterns (analyzed by SDS-PAGE electrophoresis results) have been determined at 3.1.5. Most of the target protein is present in inclusion bodies precipitated in the lysate, and the following is a specific step of washing the inclusion bodies: the cells were mixed with lysis buffer at a ratio of 1:20(g/mL) and sonicated in an ice bath (parameters were the same as in 3.1.5). Centrifuging at 12000rpm at 4 ℃ for 30min, discarding the supernatant, collecting the precipitate, and washing the inclusion body by the following steps:
(1) the first step of inclusion body cleaning is to weigh the wet weight of the sediment, add 20 times (g/mL) of washing buffer I, place the sediment for 5min at room temperature after heavy suspension, carry out ultrasonic disruption (the parameters refer to 3.1.5), wash the sediment for 1 hour under magnetic stirring at room temperature, centrifuge the sediment for 30min at 4 ℃, 12000rpm, discard the supernatant and leave the sample to obtain the sediment inclusion body.
(2) And in the second step of inclusion body cleaning, 20 times (v/v) of washing buffer solution II is added into the sediment for resuspension, ultrasonic crushing is carried out (parameters are the same as above), magnetic stirring washing is carried out for 1h at 12000rpm and 4 ℃, centrifugation is carried out for 30min, and the supernatant is discarded and a sample is reserved to obtain the sediment inclusion body.
(3) And in the third step of inclusion body cleaning, 20 times (v/v) of washing buffer solution III is added into the precipitate obtained in the step (2), the mixture is stirred uniformly and is subjected to ultrasonic disruption (the parameters are the same as the above). Washing with magnetic stirring at 12000rpm for 1h, centrifuging at 4 deg.C for 30min, discarding the supernatant and reserving the sample to obtain the precipitate inclusion body.
The supernatant and the precipitate of each washing are subjected to SDS-PAGE electrophoresis gel running to determine whether the target protein is in the precipitate, so that a renaturation experiment is performed.
2.4 renaturation of the protein of interest:
weighing the washed inclusion body, adding 20 times (g/V) of the denaturation liquid for dissolving, stirring for 15h by a magnetic stirrer, 12000rpm, centrifuging for 30min at 4 ℃ to obtain supernatant, namely the inclusion body denaturation liquid, precipitating to become cell debris and other magazines, pumping 7 times of volume of renaturation balance liquid into the denaturation liquid in a beaker by a pump with the flow rate of 0.1mL/min, and putting a magnetic stirrer into the beaker for continuous stirring until the renaturation balance liquid is completely added.
2.5 purification of the renatured target protein by a tweezer column one-step method:
(1) filling the column, filling the nickel agar gel medium into a chromatography column washed by double distilled water, avoiding shaking the column when pouring, slowly pouring along the tube wall, and ensuring that the volume of a column bed is about 7 cm. After the gel naturally precipitated, the pump was turned on, and 5 column volumes of autoclaved sterile filter-sterilized double distilled water were pumped into the column at a flow rate of about 1 mL/min.
(2) Equilibration 5 column volumes of equilibration buffer were pumped into the column to equilibrate the nickel column.
(3) And (4) centrifuging the renatured liquid, taking the supernatant, filtering the supernatant through a 0.22 mu L filter membrane, pumping the supernatant, and repeatedly pumping the effluent for 4 times to ensure that the proteins are combined in the nickel column.
(4) And (3) eluting the hybrid protein, observing an ultraviolet baseline, and eluting with an eluent containing 100mM imidazole when the baseline is not changed, wherein the volume is 5 column volumes until the ultraviolet baseline is not changed.
(5) And (3) eluting the target protein by pumping 5 column volumes of imidazole containing 300mM into a nickel column, eluting the target protein, recording the elution peak value, collecting the eluent when the value is obviously high, wherein each tube contains about 1mL, and recording the value of each tube.
(6) And when the ultraviolet numerical value is unchanged, indicating that the target protein is completely eluted, and stopping collecting the eluent. And sequentially pumping 5 column volume balance buffer solutions, high-pressure filter sterilized double distilled water and 35mL of 20% ethanol, and finally storing in a refrigerator at 4 ℃.
(7) The purified samples were analyzed by SDS-PAGE electrophoresis.
2.6 dialysis and concentration of the protein of interest:
(1) removing 15mL of protein sample purified by the imidazole nickel column in the purified protein, measuring and centrifuging through an ultrafiltration centrifugal tube for 25min at 5000g, filtering to obtain liquid volume, adding equal volume of 1 XPBS, centrifuging for three times under the same condition, and removing imidazole in the purified protein.
(2) Concentration of protein the protein solution obtained in (1) above was centrifuged continuously until the volume was concentrated to 1 mL.
2.7 quantitative analysis of concentrated protein concentration:
the concentrated recombinant IZUMO protein is determined by using a BCA protein concentration determination kit, and the specific steps are as follows: (1) and (3) preparing the BCA reagent, namely, uniformly mixing the reagent A and the reagent B according to the proportion of 50:1 for later use.
(2) The standard protein was pipetted at 0. mu.L, 1. mu.L, 2. mu.L, 4. mu.L, 8. mu.L, 12. mu.L, 16. mu.L, 20. mu.L, respectively, onto a 96-well plate. To the corresponding wells, 20. mu.L, 19. mu.L, 18. mu.L, 16. mu.L, 12. mu.L, 8. mu.L, 4. mu.L, and 0. mu.L of double distilled water were added in this order.
(3) Concentrated protein was added at 20. mu.L per well.
(4) Adding 200 mu L of BCA reagent into each well, and reacting at 37 ℃ for 30-60 min.
(5) Cooling to room temperature, comparing with blank, performing color comparison at 595nm on an enzyme labeling instrument, and calculating the concentration of the target protein according to the standard curve.
2.8 SDS-PAGE electrophoretic analysis and Western blot identification of the purified proteins:
SDS-PAGE electrophoresis was performed on the purified proteins and Western blot identification was performed, the procedures of the SDS-PAGE electrophoresis were as described above, and the results are shown in FIG. 7. Western blot identification respectively adopts an anti-His-tag mouse monoclonal antibody and a mouse anti-human IZUMO polyclonal antibody as primary antibodies, and the dilution concentration is 1: 1000; the secondary antibody is rabbit anti-mouse IgG marked by HRP, the dilution concentration is 1:1000, and the secondary antibody is finally exposed and photographed in a gel imager to analyze immunoreaction strips. The results are shown in FIGS. 8 and 9.
And thirdly, establishing and optimizing an ELISA method based on detection of the IZUMO antibody and detection of clinical samples.
3.1 basic steps of ELISA:
(1) coating: diluting the target protein by using a coating buffer solution, coating the diluted protein-containing coating solution on a 96-well enzyme label plate by using 100 mu L/well, incubating for 12h at 4 ℃, washing for 5 times by using a washing solution, slightly shaking by holding the plate by hands each time, paying attention to not spill to pollute adjacent wells, washing, throwing off the washing solution, and patting to dry;
(2) and (3) sealing: adding 250 mu L of confining liquid into each hole, incubating for 2h at 37 ℃, washing the plate for 5 times, and drying;
(3) adding a sample: adding samples, incubating for 1h at 37 ℃ with 100 mu L of each well, washing the plate for 5 times, and drying;
(4) adding horseradish peroxidase-labeled secondary antibodies: adding HRP-mouse anti-human monoclonal antibody IgG with a certain dilution concentration into each hole, incubating for 1h at 37 ℃ in each hole with 100 mu L, washing the plate and drying for 5 times;
(5) color development: adding one-step color-developing agent TMB, incubating at 37 deg.C in dark for 15min, wherein each well has a volume of 50 μ L;
(6) and (3) terminating the reaction: adding 50 mu L of ELISA stop solution into each reaction hole, and finishing color comparison within 5 minutes;
(7) color comparison: the OD value of each well at the detection wavelength of 450 nm. The schematic diagram of the indirect ELISA experiment is shown in FIG. 8.
3.2 optimal HRP-labeled murine anti-human monoclonal antibody IgG (enzyme-labeled secondary antibody) dilution concentration determination:
coating a 96-hole micro enzyme label plate with human IgG standard protein at the concentration of 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 600ng/mL and 800ng/mL, simultaneously diluting an enzyme-labeled secondary antibody at the ratio of 1:2000, 1:5000, 1:6000 and 1:8000, setting two multiple holes for each gradient, and calculating the average value. Determining the optimal horse radish peroxidase labeled mouse anti-human IgG (secondary antibody) working concentration according to a chessboard titration method, namely detecting A by an enzyme labeling instrument450Secondary antibody concentrations at values close to 1.
Human IgG standard protein coats the ELISA plate with different concentrations, HRP-labeled secondary antibodies with the dilution concentrations of 1:2000, 1:5000, 1:6000 and 1:8000 are added into each hole, and two multiple holes are arranged in each concentration gradient. A. the450The concentration corresponding to approximately 1 is the optimal dilution concentration for the secondary antibody, and the optimal working concentration for the secondary antibody is 1:5000 as shown in table 4 below.
Table 4: absorbance values of enzyme-labeled secondary antibodies of different dilutions
3.3 determining the optimal dilution concentration of the coating protein and the positive standard:
and (3) diluting the target protein by carbonate buffer solution, wherein the concentration is 0.1ng/mL, 0.25ng/mL, 0.5ng/mL, 1ng/mL, 5ng/mL and 10ng/mL in sequence, the positive standard substance is diluted by 1:25, 1:50, 1:100 and 1:200 times, the negative standard substance is also diluted by the same proportion, each concentration gradient has two multiple wells, and the average value is obtained. At the wavelength of 450, the OD value of the positive standard is about 1, the negative OD value is less than 0.2, and the ratio of the OD value of the positive standard to the negative OD value (P/N value) is maximum, so that the protein coating concentration and the optimal dilution concentration of the positive standard can be determined.
According to the absorbance result measured by a chessboard titration enzyme-labeling instrument, when the OD value of the positive standard substance is close to 1, the negative standard substance is less than 0.2, the P/N value is larger, the negative control is smaller, the optimal coating concentration is 5ng/mL, and the optimal dilution concentration of the positive standard substance is 1:100, as shown in Table 5.
Table 5: ELISA (enzyme-Linked immunosorbent assay) for detecting absorbance values of different protein coating concentrations
3.4 selection of coating liquid: two multiple holes are arranged in each concentration gradient, color development and color comparison are completed according to the indirect ELISA step, and A is measured450The value is obtained. All in one
The ELISA coating solution generally comprises four kinds of PBS, sodium carbonate buffer solution, TRIS buffer solution and normal saline (0.9% NaCl), the concentration of the target protein dissolved in the ELISA coating solution is 5ng/mL, 100 mu L (3) of positive standard substance dilution concentration is added into each hole, and the coating solution with the maximum P/N and smaller negative standard substance and blank control OD values is the optimal coating solution.
3.5 for the selection of the blocking solution:
coating the ELISA plate with the target protein with determined concentration, respectively coating the blocking solution with 3% PBS, 5% PBS, 3% PBST and 5% PBST, setting two complex holes for each concentration gradient, performing color development and color comparison according to indirect ELISA procedure, and measuring A450The value is obtained. The confining liquid with the maximum P/N and the small OD values of the negative standard substance and the blank control is the optimal confining liquid.
3.6 blocking temperature selection:
and (3) sealing for 2h under the sealing conditions of 4 ℃ overnight and 37 ℃, setting two multiple holes for each concentration gradient, finishing color development and color comparison according to an indirect ELISA step, and setting the position with the maximum P/N and the smaller OD values of the negative standard substance and the blank control as the optimal sealing temperature.
3.7 TMB color developing agent action time selection:
finishing the pre-development step according to the indirect ELISA step, wherein the development time is respectively 10min, 15min, 20min and 25min, and the detection A is carried out450The value is obtained.
3.8 repeatability evaluation of the kit for ELISA detection of IZUMO antibody:
according to the conditions, after the kit is standardized, an IZUMO standard antibody is adopted, and the repeatability of the IZUMO antibody indirect method detection kit is evaluated by respectively using the same reagent plate and reagent plates coated at different times for detection.
The same concentration of IZUMO antibody standard (1:1000 dilution) was tested in the same coated plate and 10 replicate wells were made, and the mean of replicate wells was used to calculate the coefficient of variation (CV1) with a CV value of less than 0.018 as shown in Table 6. In addition, 5 plates of the kit are coated at different times for detecting the IZUMO antibody with the dilution of 1:1000, the variation coefficient (CV2) is calculated by the mean value of the 5 plates, and the result shows that the variation coefficients of the detection values among the different plates are all less than 0.031, which indicates that the kit for detecting the IZUMO antibody by the indirect ELISA method standardized according to the conditions has good repeatability.
TABLE 6 results of the repeatability tests
3.9 clinical specimen detection:
the above optimized ELISA method is used for detecting the collected 30 normal pregnant woman physical examination serum and 65 clinical confirmed pregnant woman patient serum, and the result shows that: the OD value of the antisperm antibody in the peripheral blood serum of healthy pregnant women is (0.01303 +/-0.004803), 6 cases of 65 infertility female patients have P/N values (the ratio of the OD value to the average OD value of a normal control group) of more than 3, the pregnant women are considered to be positive, and the OD values of 30 normal pregnant women and 65 clinically confirmed infertility female patients are shown in a table 7.
TABLE 765 test results of clinical samples
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Amino acid sequence listing
<110> Guangdong university of medical science
<120> antigen for detecting sperm antibody, expression vector thereof, kit thereof and indirect ELISA detection method
<160> 2
<210> SEQ ID NO: 1
<211>94
<212> PRT
<213> peptide B, PB
<400> SEQ ID NO: 1
MERNVEVPQM EDMILDCELN WHQASEGLTD YSFYRVWGNN TETLVSKGKE
ATLTKPMVGP EDAGSYRCEL GSVNSSPATI INFHVTVLHH HHHH
Gene sequence table
<110> Guangdong university of medical science
<120> antigen for detecting sperm antibody, expression vector thereof, kit thereof and indirect ELISA detection method
<160> 2
<210> SEQ ID NO: 2
<211>285
<212> DNA
<213> peptide B, PB
<400> SEQ ID NO: 2
ATGGAACGTA ATGTGGAAGT CCCGCAAATG GAAGATATGA TTCTGGATTG TGAACTGAAC TGGCACCAAG CCTCTGAAGG TCTGACGGAT TATAGCTTTT ACCGTGTGTG GGGCAACAAT ACCGAAACGC TGGTTTCTAA GGGTAAAGAA GCGACCCTGA CGAAACCGAT GGTCGGCCCG GAAGATGCCG GTAGTTATCG CTGCGAACTG GGCTCGGTGA ACTCCTCCCC GGCAACGATT ATCAACTTCC ATGTGACGGT CCTGCATCAT CACCATCACC ACTAA
Claims (6)
1. An antigen for detecting sperm antibodies, comprising: the antigen for detecting the sperm antibody is polypeptide encoded by a gene fragment of a PB functional region of a recombined IZUMO gene, the amino acid sequence of the polypeptide is a sequence shown by SEQ ID NO. 1, and the gene sequence of the polypeptide is a sequence shown by SEQ ID NO. 2.
2. An expression vector of a gene encoding an antigen for detecting sperm antibody according to claim 1, wherein: the expression vector is pET30 a-IZUMO-His.
3. The method of constructing an expression vector of a gene encoding an antigen for detecting a sperm antibody according to claim 2, wherein: the method comprises the following steps:
step one, synthesizing a gene fragment of a PB functional region of a recombinant IZUMO gene: obtaining a PB gene sequence by adopting a gene synthesis mode, and inserting a His label at the 3' end to obtain a gene segment with a sequence shown as SEQ ID NO. 2;
step two, PB gene fragment amplification: and (4) carrying out PCR amplification by taking the gene fragment obtained in the step one as a template to obtain the expression vector of the antigen for detecting the sperm antibody.
4. The method for constructing an expression vector for detecting an antigen of a sperm antibody according to claim 3, wherein: in the second step, the PCR amplification conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 40s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 45s, 30 cycles, and extension at 72 ℃ for 10 min.
5. An ELISA kit for detecting an anti-sperm antibody, characterized in that: comprises the following components: the detachable elisa plate coated by the antigen of claim 1, a positive standard, a negative control, an anti-antibody labeled by horseradish peroxidase, a developing solution and a stop solution.
6. The ELISA kit for antisperm antibody detection according to claim 5, characterized in that: the detachable enzyme label plate is a 96-hole corning detachable enzyme label plate; the positive standard substance is a rabbit anti-human IZUMO recombinant sperm antigen PB functional region polypeptide antibody, and the negative control substance is virgin serum.
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| JP2010187556A (en) * | 2009-02-16 | 2010-09-02 | Osaka Univ | Pregnancy-diagnosing method, pregnancy-diagnosing kit, polynucleotide, polypeptide, and antibody |
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