Endometrial specific fusion protein antigen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and discloses an endometrial specific fusion protein antigen as well as a preparation method and application thereof. The amino acid sequence of the novel endometrial specific fusion protein antigen provided by the invention is shown as SEQ ID NO.1, and the novel endometrial specific fusion protein antigen comprises a linear antigen PAEP protein fragment and a Nidogen-1 protein fragment which are connected front and back, the antigen sequentially contains 132 amino acids from the 19th site to the 150th site of PAEP protein from the N-terminal to the C-terminal and 231 amino acids from the 520th site to the 750th site of Napogen-1 protein from the N-terminal from the N-terminal to the C-terminal. According to the preparation method provided by the invention, the endometrial specific fusion protein antigen is massively expressed and purified through an escherichia coli expression system to obtain the diagnostic antigen, and the requirements of development and application of the diagnostic kit are met. The fusion protein is good in specificity and high in sensitivity, the detection specificity and sensitivity are obviously improved, and missing detection is effectively prevented.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an endometrium-specific fusion protein antigen and a preparation method and application thereof. Background Art

[0002] Anti-endometrial antibodies (EMAb) are a common cause of female infertility and are common in patients with endometriosis. Anti-endometrial antibodies are autoantibodies that use the endometrium as a target antigen and induce a series of immune responses. Reports have shown that the positive rate of anti-endometrial antibodies in the blood of women with endometriosis and infertility is significantly higher than that of normal controls. In the serum of endometriosis, the detection rate of EMAb can reach 70%-80%.

[0003] Endometriosis (EMS) is a complex, common gynecological disease that affects about 5% to 15% of women of childbearing age. It is closely related to dysmenorrhea, pelvic pain, and decreased fertility, and its pathogenesis is very complex. The diagnosis of EMS mainly relies on laparotomy exploration and laparoscopy. Electron microscopy shows endometrial dysplasia, vacuoles in endometrial glands and basement membranes, and a decreased ratio of ciliated to non-ciliated cells. In order to seek a non-invasive detection method, endometrial antibodies (EMAg) were purified from the secretory endometrium of normal women, and an ELISA detection method for anti-endometrial antibodies was established. This method was used for the early diagnosis of EMS.

[0004] There are two aspects to the production of anti-endometrial antibodies: on the one hand, it is because of the ectopic endometrium stimulating the system; on the other hand, it is because of the body's immune system disorder (autoimmune deficiency). Anti-endometrial antibodies target antigens such as progesterone-dependent protein (PAEP), S100P and Nidogen-1.

[0005] Ectopic endometrium is antigenic and can induce the body to produce anti-endometrial antibodies. When antigens and antibodies combine, they can be deposited in the endometrium and ectopic lesions, destroying the endometrial structure by activating complement, affecting endometrial development and endocrine function. This pathological damage to the endometrium can lead to a decrease in the level of glycogen that nourishes the embryo and an increase in the level of immune complexes, resulting in implantation failure of the fertilized egg and poor embryo development, thereby causing infertility or miscarriage. During artificial abortion and curettage, the embryonic sac may also serve as an antigen to stimulate the body to produce antibodies. When a woman's endometrium is inflamed, it may also be converted into an antigen. Therefore, the detection of anti-endometrial antibodies is of great significance for diagnosis and treatment.

[0006] Currently, the clinical detection of EMAb mainly uses the enzyme-linked immunosorbent assay (ELISA) method, which is fast, has low experimental requirements, does not require sterile operation, can quickly and high-throughput detect a large number of samples, is also relatively sensitive and reliable, and has been widely used in different pathogen detection.

[0007] The positive rates of infertility antibodies reported in the literature vary greatly. One of the important reasons is that the current method for preparing endometrial antigens is still relatively rough. Endometrial antigens need to be extracted from tissue homogenates, which is not only difficult to obtain tissue sources but also difficult to separate high-purity proteins. In addition, the antigen composition is complex, the specificity is poor, and the differences between test batches are large, resulting in poor consistency in the test results of various laboratories. Therefore, how to develop endometrial antigens with higher purity, stronger specificity, and high consistency between batches is a problem that needs to be solved at present.

[0008] Therefore, the prior art needs to be further improved. Summary of the invention

[0009] In response to the above problems, the present invention provides an endometrium-specific fusion protein antigen and a preparation method and application thereof. The endometrium-specific fusion protein antigen has strong specificity and high affinity. By adopting a high-efficiency expression prokaryotic expression system, the yield and purity of the endometrium-specific fusion protein antigen are greatly improved, laying a foundation for studying the biological activity of human endometrial antigens.

[0010] To solve the above problems, this application provides the following technical solutions: In the first aspect, the present application provides an endometrium-specific fusion protein antigen, which includes a linear antigen PAEP protein fragment and a Nidogen-1 protein fragment connected in front and behind, wherein the linear antigen PAEP protein fragment is 132 amino acids from the 19th to the 150th position of the N-terminus, and the Nidogen-1 protein is 231 amino acids from the 520th to the 750th position of the N-terminus, and the amino acid sequence of the endometrium-specific fusion protein antigen is SEQ ID NO: 1.

[0011] Specifically, the sequence of the endometrium-specific fusion protein antigen contains, from N-terminus to C-terminus, 132 amino acids of PAEP protein from N-terminus 19 to 150 and 231 amino acids of Nidogen-1 protein from N-terminus 520 to 750. There is no connecting peptide between the two sequences.

[0012] In a second aspect, the present application also provides a coding gene for the aforementioned endometrial specific fusion protein antigen, the nucleotide sequence of which is shown in SEQ ID NO: 2. The sequence is a sequence after codon optimization based on the E. coli expression system, does not contain rare codons of E. coli, and is suitable for efficient expression in the E. coli expression system.

[0013] In other cases, in order to express the endometrium-specific fusion protein antigen with the above amino acid sequence as shown in SEQ ID NO: 1 in other prokaryotic or eukaryotic systems, the gene sequence can be optimized accordingly based on conventional technical means in the art to obtain the corresponding coding sequence.

[0014] Therefore, the endometrium-specific fusion protein antigen protected in the present application is not limited to the encoding gene sequence as shown in SEQ ID NO: 2.

[0015] In a third aspect, the present application also provides a recombinant expression vector, which is formed by the recombination of an expression vector and the coding gene of the aforementioned endometrium-specific fusion protein antigen.

[0016] In a fourth aspect, the present application also provides a recombinant expression vector, which is a pET series expression vector, such as PET-32a, pET-28a or pET-43.1a.

[0017] In other cases, the endometrium-specific fusion protein antigen encoding gene sequence can be optimized accordingly according to the selected expression system and based on conventional technical means in the art, and the expression vector can use an expression vector corresponding to the expression system (other prokaryotic or eukaryotic expression systems).

[0018] In a fifth aspect, the present application also provides a host cell for transforming the aforementioned recombinant expression vector.

[0019] Optionally, the recombinant engineered bacteria are Escherichia coli BL21 (DE3) competent cells, Escherichia coli Rosseta or other Escherichia coli mutant expression strains, etc.

[0020] In other cases, based on the type of expression vector of the selected prokaryotic or eukaryotic expression system, the host bacteria can be Escherichia coli Rosseta or other Escherichia coli mutant expression strains, Pichia pastoris, etc. that match the expression vector.

[0021] By using recombinant endometrial antigen, which is secreted and expressed by Escherichia coli, the antigen protein has high purity, which can reduce the inevitable interference during natural extraction, optimize the recombinant endometrial antigen purification process, minimize the differences in test results between different batches, and make the results more reliable.

[0022] In a sixth aspect, the present application further provides a method for preparing the aforementioned endometrium-specific fusion protein antigen, which comprises the following steps: (1) cloning the nucleotide sequence of the aforementioned endometrial-specific fusion protein antigen encoding gene into an expression vector to obtain a recombinant expression vector; (2) Transforming the recombinant vector into the host bacteria and screening to obtain the recombinant engineered bacteria; (3) The recombinant engineered bacteria are cultured and induced to express, and then purified by affinity chromatography to obtain the recombinant antigen.

[0023] In a seventh aspect, the present application also provides an antibody detection kit, wherein the diagnostic antigen used is the aforementioned endometrium-specific fusion protein antigen.

[0024] Optionally, the detection kit is a fluorescent immunochromatography kit, a colloidal gold detection kit or an enzyme-linked immunosorbent assay kit.

[0025] Optionally, the endometrium-specific fusion protein antibody detection kit further comprises: a detection plate coated with the aforementioned endometrium-specific fusion protein antigen.

[0026] Optionally, the kit further comprises: antibodies, enzyme markers, substrates and reference standards.

[0027] The endometrium-specific fusion protein antigen can be used to detect anti-endometrial antibodies. Those skilled in the art can prepare a detection kit using the endometrium-specific fusion protein antigen according to the disclosure of the present invention and related common sense, and use it to quickly detect anti-endometrial antibodies, which is of great significance in diagnosing female infertility or miscarriage.

[0028] The present invention has the following beneficial effects: 1. The present invention provides an endometrium-specific fusion protein antigen and a preparation method thereof. The endometrium-specific fusion protein antigen innovatively selects two preferred specific peptide segments, PAEP (19aa-150aa) and Nidogen-1 (520aa-750aa), for fusion, and uses the fusion protein as a diagnostic antigen, which significantly improves the potency and sensitivity of the fusion protein, thereby increasing the potency of the protein from the source, reducing the amount of antigen used in the diagnostic kit, and increasing the expression amount by optimizing codons, thereby increasing economic benefits.

[0029] 2. The endometrium-specific fusion protein antigen provided in the present application can be industrially produced through an E. coli expression system. The expression level of the antigen is increased by optimizing codons, and the conditions are controllable, which greatly reduces the production cost of the antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 for the analysis of PAEP antigenic sites using bioinformatics software; Figure 2 for the analysis of Nidogen-1 antigenic sites using bioinformatics software; Figure 3 It is the double enzyme digestion identification diagram of the recombinant vector; wherein, lane 1 is the marker, and lanes 2 and 3 are the double enzyme digestion results of the recombinant vector respectively; Figure 4 This is a diagram for identifying the induction of expression of recombinant protein; lane 1 is the result without induction, and lanes 2 to 5 are the results of induction. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0032] Example 1 Preparation of endometrial antigen protein 1.1 Construction of endometrial antigen protein expression vector (1) Screening of dominant epitopes The endometrial antigen sites were analyzed by online software, and specific linear antigens PAEP protein (19aa-150aa) and Nidogen-1 protein (520aa-750aa) were screened out. The analysis results of PAEP protein and Nidogen-1 protein were as follows: Figure 1 and Figure 2 shown.

[0033] The above endometrial specific fusion protein antigen encoding gene sequence was codon optimized and then synthesized into a nucleotide sequence (Sangon Biotechnology Co., Ltd.), and the nucleotide sequence is shown in SEQ ID NO.2.

[0034] Among them, the PAEP protein (19aa-150aa) is located in the 132 amino acids from the 19th to the 150th N-terminal of the PAEP protein, and the Nidogen-1 protein (520aa-750aa) is located in the 231 amino acids from the 520th to the 750th N-terminal of the Nidogen-1 protein. There is no connecting peptide between the two segments.

[0035] (2) Construction of recombinant vector EcoRⅠ and Hind Ⅲ were used to double-digest the specific nucleotide sequence synthesized in the above steps and the expression vector PET32A. The enzyme digestion reaction system was: EcoRⅠ (1ul), Hind Ⅲ (1ul), 10×QuickCut buffer (3ul), synthetic gene (20ul), ddH2O (5ul), 37℃ enzyme digestion for 1h, and 1% agarose gel electrophoresis was performed after the reaction, and the target gene and expression vector were recovered by gel. The specific nucleotide sequence and expression vector recovered by gel were added to the ligation reaction system as required. The ligation reaction system was: specific nucleotide (2ul), linearized vector (2ul), T4 DNA Ligase (1ul), 10×T4 DNALigase Buffer (1ul), 16℃ connection for 2h, and the recombinant expression vector was obtained after connection.

[0036] 1.2 Obtaining endometrial antigen-expressing engineered bacteria and expressing and purifying fusion protein antigens (1) Transformation of ligation products Add the ligated product to 50 μl BL21 competent cells, mix gently, and place on ice for 30 min; heat shock at 42 °C for 90 s, and quickly place on ice for 3 min; add 500 μl LB liquid culture medium, incubate at 37 °C with shaking at 180 rpm for 1 h; spread the transformed bacterial solution on an LB plate containing antibiotics and culture overnight at 37 °C.

[0037] (2) Obtaining engineered bacteria The positive clones were selected and inoculated into 2 ml LB medium containing ampicillin for culture. The recombinant plasmid was extracted and double enzyme digestion was performed. The samples after enzyme digestion were subjected to 1% agarose gel electrophoresis for preliminary identification. The enzyme digestion identification diagram is shown in Figure 3 The strains that are initially verified to be correct are then sent to the company for sequencing, and the correct strains are further identified as engineered bacteria, which are then kept for future use.

[0038] (3) Identification of recombinant antigen expression Pick a single colony growing on the plate and inoculate it into a test tube containing 2 ml LB medium, and culture it at 37°C 220rpm overnight to activate the seeds. The next day, take 20μl of the overnight cultured bacterial solution to a sterilized 2ml LB medium containing antibiotics, and culture it at 37°C, 220rpm until the OD value is about 0.6-0.8, add a final concentration of 1mM IPTG, and induce expression for 5h. The induction conditions are 28°C, 220rpm, and 5h. Centrifuge at 4000rpm for 10min at room temperature, discard the supernatant, and resuspend the precipitated bacteria in 400μl 10mM PBS. Take a sample for SDS-PAGE to identify the expression of the target protein in E. coli BL21 (see Figure 4). The cloned strain expressing the correct gene was preserved for later use.

[0039] (4) Inducible expression of recombinant protein Take out the strain stored at -70℃, streak it on an LB plate, and culture it in a 37℃ incubator overnight until a single colony grows. Pick a single colony, add 10ml LB to a 50ml centrifuge tube, and culture it overnight at 37℃ and 220r / min with ventilation to obtain an overnight culture. Take 5ml of the overnight culture and add it to a 1000ml conical flask containing 500ml of culture medium, and culture it at 37℃ and 220rpm until the OD600 is about 0.6-0.8. Add IPTG at a final concentration of 0.1mM, 220rpm, 28℃, and induce for 4.5-5h. Centrifuge at 4℃ and 9000rpm for 15min, collect the precipitate, and store it at -20℃ for later use.

[0040] (5) Protein purification Each 1g of bacterial pellet was fully resuspended with 20mL Washbuffer (20mM imidazole + 50mM NaH2PO4·2H2O + 500mMNaCl, Ph7.6), and then ultrasonically disrupted. The bacterial disruption solution was centrifuged at 4°C, 9000r / min for 10min, and the supernatant was collected to obtain the disrupted bacterial supernatant. The disrupted bacterial supernatant was filtered with a 0.45-μm microporous filter membrane. Take 4mL of Ni-NTA agarose gel FF pre-packed column, equilibrate it with 50mL of Washbuffer, then load the sample, collect the effluent, and load the effluent again to allow the protein sample to fully bind to the gel, wash away the unabsorbed sample with 50mL of Washbuffer, elute the target protein with 15mL of Elutionbuffer (500mM imidazole + 50mM NaH2PO4·2H2O + 500mM NaCl, Ph7.6), rinse the column with pure water, fill it with 20% ethanol, and seal it for next use.

[0041] Example 2 Preparation of polyclonal antiserum against endometrial specific fusion protein antigen 1. Experimental methods Endometrial specific fusion protein was used as an immunogen to immunize 5 New Zealand rabbits, each of which was immunized with 200ug antigen, and the immunization method was multiple points on the back. The first immunization used complete Freund's adjuvant and fusion protein mixed and emulsified; two weeks later, the second immunization used incomplete Freund's adjuvant and fusion protein mixed and emulsified, and the immunization method was multiple points on the back; three days later, the third immunization was carried out, using 100ug of fusion protein and the same volume of 0.01M PBS (PH7.4) mixed, and the immunization method was intraperitoneal immunization; three days later, the same immunization procedure was used for immunization again, and the antibody titer was detected by indirect ELISA method.

[0042] 2. Experimental results and analysis It was detected that the rabbit antiserum titer was as high as more than 10,000, indicating that the endometrium-specific fusion protein antigen prepared by the present invention has good antigenicity and specificity.

[0043] Example 3 Preparation of a detection kit for anti-endometrial antibodies 1. Preparation method of the kit The anti-endometrial antibody detection kit comprises: a detection plate coated with endometrial specific fusion protein antigen, an enzyme-labeled antibody, a substrate and a reference standard.

[0044] The detection principle of the above detection kit is as follows: the endometrial-specific fusion protein antigen is bound to the surface of a solid phase carrier and its immune activity is maintained, the antibody to be tested is added, and the specific binding of the antigen and the antibody is utilized to detect the anti-endometrial antibodies in human serum.

[0045] 2. Detection method of the kit The purified recombinant protein was diluted with carbonate buffer (50mM, pH9.6) to a protein content of 1μg / ml, and 100ng / well of protein was coated on the ELISA plate, incubated at 4℃ overnight, washed, added with 200ul / well of 10% calf serum in PBS, blocked in a 37℃ water bath for 2h, washed and set aside at 4℃; added the sample to be tested to react with the coated antigen, incubated at 37℃ for 1 hour, washed, added freshly diluted enzyme-labeled antibody to each reaction well, incubated at 37℃ for 0.5-1 hour, washed, added substrate solution for color development, and the OD value was determined using an enzyme reader at a wavelength of 450nm.

[0046] According to the above method, the serum samples of 100 clinically positive patients were tested using the above kit, and the serum samples of 100 healthy controls were simultaneously tested.

[0047] 3. Test results and analysis The test results are as follows: 98 of the 100 healthy control sera were negative and the remaining 2 were positive. 99 of the 100 positive sera were positive and the remaining 1 was negative. This result shows that the sensitivity of the kit of the present invention is 99% and the specificity is 98%. It has good sensitivity and specificity.

[0048] Example 4 Comparative analysis of the experimental group and the control group 1. Experimental setup This example is to compare the effects of the endometrium-specific fusion protein antigen of the present application with the antigens of other control groups, and the following experimental groups and control groups are set up respectively.

[0049] The experimental group used the method of Example 1 to prepare the endometrial specific fusion protein antigen, while the antigens prepared in the comparative groups 1 and 2 were operated as follows: (1) The amino acid sequence used in the comparative group 1 is 132 amino acids from the 19th to the 150th position of the PAEP protein from the N-terminus, and the sequence is shown in SEQ ID NO. 3. Prokaryotic expression was performed according to the steps of Example 1-2, and other treatments were consistent with the experimental group.

[0050] (2) The amino acid sequence used in control group 2 is 231 amino acids from the 520th to the 750th position of the N-terminal of Nidogen-1 protein, and the sequence is as shown in SEQ ID NO. 4. Prokaryotic expression was performed according to the steps of Example 1-2, and other treatments were consistent with the experimental group.

[0051] (3) The amino acid sequence used in control group 3 is 102 amino acids from the 19th to the 120th position of the PAEP protein and 141 amino acids from the 520th to the 660th position of the Nidogen-1 protein from the N-terminus. The sequence is as shown in SEQ ID NO. 5. Prokaryotic expression was performed according to the steps of Example 1-2. Other treatments were consistent with the experimental group.

[0052] (4) The amino acid sequence used in control group 4 is 85 amino acids from the 66th to the 150th position of the PAEP protein and 90 amino acids from the 661st to the 750th position of the Nidogen-1 protein from the N-terminus. The sequence is as shown in SEQ ID NO. 6. Prokaryotic expression was performed according to the steps of Example 1-2. Other treatments were consistent with the experimental group.

[0053] The experimental group and the control group are set up as shown in Table 1: Table 1 Comparison of antigen sequence settings and experimental results in each group

[0054] 2. Detection method: The recombinant antigens prepared in the experimental group and the control group were compared and analyzed by enzyme-linked immunosorbent assay (ELISA) The control group was subjected to prokaryotic expression according to the steps of Example 1. This experiment used the experimental group and the control group to perform the following tests: The recombinant antigen was used as a coating antigen for the gold-label chromatography platform to detect endometrial antibodies. The purified recombinant protein was diluted with carbonate buffer (50mM, pH9.6) and coated with 100ng / well of the enzyme-linked plate and incubated overnight at 4°C. After washing, 200ul / well of PBS containing 10% calf serum was added, and the plate was blocked in a 37°C water bath for 2h. After washing, it was kept at 4°C for use. 1,000 clinical positive samples and 100 healthy physical examination samples were tested by indirect enzyme-linked immunosorbent assay. Table 2 Experimental data list

[0055] 3. Experimental results and analysis From the results in Table 2, it can be seen that the sensitivity of the recombinant antigen in the experimental group of the present invention for clinical positive samples is 8.2% higher than that in the control group 1 and 5.2% higher than that in the control group 2; the specificity of the healthy physical examination samples is 6% higher than that in the control group 1 and 8% higher than that in the control group 2. Compared with the control groups 3 and 4, the detection specificity and sensitivity of the experimental group for the samples are also significantly improved. The above results show that the fusion protein antigen of the present application scheme is a clever fusion of the two preferred fragments of PAEP (19aa-150aa) and Nidogen-1 (520aa-750aa), which greatly improves the specificity and sensitivity of the recombinant antigen. The effect cannot be achieved by selecting only one of the above two fragments or a simple fragment, and the effect is unexpected.

[0056] In summary, compared with the control group, the recombinant antigen provided by the present invention has better sensitivity and specificity, and can be further applied to clinical detection.

[0057] The above contents are merely examples and explanations of the concept of the present invention. It is understandable that a person skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the scope of protection of the claims attached to the present invention.

Claims

1. An endometrium-specific fusion protein antigen, characterized in that: It includes a linear antigen PAEP protein fragment and a Nidogen-1 protein fragment connected in front and back, wherein the linear antigen PAEP protein fragment is 132 amino acids from the 19th to the 150th position of the N-terminus, and the Nidogen-1 protein is 231 amino acids from the 520th to the 750th position of the N-terminus. The amino acid sequence of the endometrium-specific fusion protein antigen is SEQ ID NO:

1.

2. A gene encoding an endometrial-specific fusion protein antigen as claimed in claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

2.

3. A recombinant expression vector, characterized in that: It is formed by recombination of an expression vector and the gene encoding the endometrial specific fusion protein antigen as claimed in claim 2.

4. The recombinant expression vector according to claim 3, characterized in that: The expression vector is a pET series expression vector.

5. A host cell transformed with the recombinant expression vector according to claim 3.

6. A method for preparing the endometrial specific fusion protein antigen according to claim 1, characterized in that: The following steps are involved: (1) cloning the nucleotide sequence of the endometrial-specific fusion protein antigen encoding gene as described in claim 2 into an expression vector to obtain a recombinant expression vector; (2) Transforming the recombinant vector into the host bacteria and screening to obtain the recombinant engineered bacteria; (3) The recombinant engineered bacteria are cultured and induced to express, and then purified by affinity chromatography to obtain the endometrial-specific fusion protein antigen.

7. A detection kit for anti-endometrial antibodies, characterized in that: The diagnostic antigen used is the endometrium-specific fusion protein antigen as claimed in claim 1.

8. The antibody detection kit according to claim 7, characterized in that: The detection kit is a fluorescent immunochromatography kit, a colloidal gold detection kit or an enzyme-linked immunosorbent assay kit.

9. The endometrium-specific fusion protein antibody detection kit according to claim 8, characterized in that: include: A detection plate coated with the endometrium-specific fusion protein antigen according to claim 1.

Images