Application of recombinant human testis specific antigen in preparation of infertility diagnosis medicine or contraceptive medicine

Abstract

The invention provides application of a recombinant human testis specific antigen in preparation of infertility diagnosis drugs or contraceptive drugs. The amino acid sequence of the antigen is shown in SEQ ID NO: 1. The recombinant human testis specific antigen is obtained by adopting a gene recombination expression technology to construct prokaryotic expression plasmids, transfecting host bacteria and inducing expression of target protein, the recombinant human testis specific antigen can be used for preparing contraceptive vaccines or immune infertility diagnostic kits, and the antigen has the specific characteristic of causing immune infertility and can be used for preparing the immune infertility diagnostic kits. The preparation method is simple and efficient, is worthy of popularization and application, and has a wide application prospect and a huge market value.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to the application of recombinant human testis-specific antigens in the preparation of infertility diagnostic drugs or contraceptive drugs. Background Art

[0002] The gonads, germ cells and reproductive hormones of humans and mammals are all antigenic and can cause immune responses in the body, affecting the occurrence and development of gametes, fertilization, blastocyst implantation, embryonic development, and delivery in the reproductive process. During gametogenesis, sperm and eggs express some specific proteins, which can serve as antigens to stimulate the body's immune system and cause immune responses. The antibodies produced by the immune response bind to the corresponding gamete antigens, which will inhibit the function of gametes and block fertilization.

[0003] In the process of studying infertility caused by immune mechanisms, people have noticed that many patients have anti-sperm antibodies in their bodies. A large number of studies have confirmed that functional defects of certain sperm surface proteins will affect the normal maturation, capacitation, acrosome reaction and sperm-egg binding of sperm, leading to infertility. With the rapid development of the economy and the further deterioration of the natural ecological environment, more and more married couples of childbearing age are troubled by infertility problems. Couples of childbearing age cannot have children for various reasons, and the proportion seems to be on the rise. This includes immune infertility caused by anti-sperm antibodies (ASA) induced by sperm antigens in the body.

[0004] Studies have shown that there are many types of human sperm antigens, which are located in multiple parts of sperm, but not all homologous antigens of ASA have an effect on fertility. Some antigens are not involved in the reproductive process, and the antibody binding region of some antigens is different from the functional region, and the antigen has no effect on its function after binding to ASA. Therefore, accurately distinguishing antibodies to infertility-related sperm proteins is of great significance for the diagnosis of clinical infertility causes and antigen neutralization treatment. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide the use of recombinant human testis-specific antigen in the preparation of infertility diagnostic drugs or contraceptive drugs. The present invention constructs a prokaryotic expression plasmid by adopting gene recombination expression technology to obtain the recombinant human testis-specific antigen, which can be used to prepare contraceptive vaccines or immune infertility diagnostic kits. The antigen has a specific characteristic of causing immune infertility. The preparation method of the antigen is simple and efficient, worthy of promotion and application, and has broad application prospects and huge market value.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides use of a recombinant human testis-specific antigen in the preparation of an infertility diagnostic drug or a contraceptive drug, wherein the amino acid sequence of the antigen includes that shown in SEQ ID NO:1.

[0008] In the detection and treatment of infertility diseases, the positive results of ordinary anti-sperm antibody detection kits have little clinical reference value, and only negative results have certain reference value. This is a consensus in clinical diagnosis and treatment and a problem that needs to be solved urgently. Using infertility-related sperm proteins as detection antigens to accurately detect the corresponding ASA can solve this specificity problem well to meet this clinical demand, but currently only applied research has been carried out on sperm-specific antigens of animals such as mice, and there has been no further in-depth research on human sperm-specific antigens. The present invention directly obtains the relevant antigen encoding gene from human testicular tissue for the first time through reverse transcription technology, and then obtains the corresponding antigen (TSA-1), which can be used for the development of relevant detection kits and the development of contraceptive vaccines.

[0009] In a second aspect, the present invention provides a nucleic acid molecule encoding the recombinant human testis-specific antigen described in the first aspect.

[0010] Preferably, the nucleotide sequence of the nucleic acid molecule includes that shown in SEQ ID NO:2.

[0011] In a third aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the second aspect.

[0012] Preferably, the expression vector is obtained by connecting the nucleic acid molecule described in the second aspect to the pGEX-4T-2 vector.

[0013] In a fourth aspect, the present invention provides a host cell, wherein the host cell contains at least one copy of the expression vector described in the third aspect; or the nucleic acid molecule described in the second aspect is integrated into the genome of the host cell.

[0014] Preferably, the host cell is Escherichia coli.

[0015] In a fifth aspect, the present invention provides a method for preparing a recombinant human testis-specific antigen, the preparation method comprising:

[0016] (1) RNA was extracted from human testicular tissue by reverse transcription PCR amplification to obtain a gene encoding a human testicular specific antigen, wherein the upstream primer sequence of the reverse transcription PCR amplification is shown in SEQ ID NO: 3; and the downstream primer sequence of the amplification is shown in SEQ ID NO: 4;

[0017] (2) constructing the obtained coding gene on the expression vector pGEX-4T-2 and transferring it into a competent host bacterium to obtain a recombinant expression engineering bacterium;

[0018] (3) culturing the recombinant expression engineering bacteria, inducing expression, and then purifying to obtain the recombinant human testis-specific antigen.

[0019] Preferably, the step of constructing the coding gene comprises: performing double digestion on the obtained coding gene fragment and the expression vector pGEX-4T-2 with restriction endonucleases respectively, purifying the obtained coding gene fragment and the open expression vector after digestion, and connecting the coding gene fragment and the open expression vector to obtain a recombinant expression vector.

[0020] Preferably, the restriction endonucleases include SmaI and SalI.

[0021] Preferably, the host bacteria is selected from: Escherichia coli TG1 or Escherichia coli Ecoli JM109.

[0022] Preferably, the transfer method includes: chemical transfection or electroporation.

[0023] In the present invention, the recombinant expression engineering bacteria are cultured, induced to express, and then purified to obtain the recombinant human testis-specific antigen.

[0024] In the present invention, the steps of inducing expression and purification include: expanding the culture of the recombinant expression engineering bacteria, inducing the expression of rhTSA-1 with a GST tag at a final concentration of 0.9-1 mmol / L IPTG, 220-250 rpm for 4-5 hours, collecting the bacterial precipitate at 11000-12000g, 0-4°C for 9-10 minutes, resuspending the bacterial cells in pH7.3 PBS (containing 5 mmol / L DTT), ultrasonically disrupting the bacterial cells, 11000-12000g, 0-4°C for 15-20 minutes to collect the supernatant, loading the supernatant on a Glutathione sepharose 4Fast Flow affinity column, eluting with a pH7.3 PBS (containing 5 mmol / L DTT) equilibrium solution, and finally eluting with a 45-50 mmol / L pH8.0 Tris-HCl (containing 10 mmol / L reduced glutathione) eluent to collect the target peak.

[0025] The present invention adopts RT-PCR method to amplify the cDNA of human TSA-1 from the total RNA of human testicular tissue, clones it into the expression vector pGEX-4T-2, and constructs the recombinant prokaryotic expression plasmid pGEX / TSA-1 of TSA-1. After enzyme digestion and sequencing identification, the recombinant plasmid is transformed into Escherichia coli JM109 and induced to express in Escherichia coli to obtain the recombinant human testicular specific antigen. After SDS-PAGE electrophoresis, the results show that the relative molecular weight of the expression product is consistent with the expected result.

[0026] In a sixth aspect, the present invention provides the use of the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, or the recombinant human testis-specific antigen described in the fifth aspect in the preparation of infertility diagnostic drugs or contraceptive drugs.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention provides an ideal target site for designing vaccines for sperm antigens associated with infertility. The candidate sperm-related antigens for ideal contraceptive vaccines are preferably tissue-expression-specific proteins, and TSA-1 protein is specifically expressed only in human testicles.

[0029] (2) The present invention successfully cloned the coding gene of human TSA-1, induced the expression of recombinant human TSA-1, and established a corresponding purification process. The purity of the prepared rhTSA-1 exceeded 98%, which laid a certain foundation for the diagnosis and treatment of immune infertility and the development of contraceptive vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the electrophoresis result of the RT-PCR amplification product of human TSA-1.

[0031] Figure 2 This is the result of enzyme digestion identification of the recombinant plasmid pGEX / TSA-1.

[0032] Figure 3 This is the result of the induced expression of rhTSA-1. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0034] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] Example 1

[0036] 1. Reagents used in the experiment

[0037] Human testicular tissue was obtained from surgery in our hospital. The total RNA extraction reagent Trizol was purchased from Invitrogen. The reverse transcription kit and SalⅠ and SmaⅠ were products of Promega. Taq DNA polymerase and T4 DNA ligase were products of Takara Biotechnology (Dalian) Co., Ltd. PCR product recovery, gel recovery and plasmid extraction kits were products of QIAGEN. Glutathione sepharose 4Fast Flow filler was purchased from Amersham. The vector pGEX-4T-2 was purchased from Clotech, and Ecoli JM109 was stored in our laboratory. The amplification primers were designed based on the gene sequence of TSA-1 in GenBank.

[0038] 2. RT-PCR of human TSA-1

[0039] According to the kit instructions, total RNA was extracted from human testicular tissue using Trizol, reverse transcribed into the first strand of cDNA, and the first strand of cDNA was used as a template to amplify the human TSA-1 coding gene using the upstream primer (SEQ ID NO: 3) 5'-CCCCCGGGATGAGAAAATGTTGTCC-3' and the downstream primer (SEQ ID NO: 4) 5'-CCGTCGACTTAAATACCACTTTTTC-3'. The upstream primer contained the start codon ATG and the introduced restriction site SmaⅠ, and the downstream primer contained the stop codon TAA and the introduced restriction site SalⅠ.

[0040] 3. Construction and identification of human TSA-1 expression plasmid

[0041] The RT-PCR product was recovered by a recovery kit, and the pGEX-4T-2 vector was respectively digested with SalⅠ and SmaⅠ in a 20μL reaction volume, placed in a 37℃ water bath for 5h, and electrophoresed with 10g / L agarose gel, and the target fragment was recovered by gel cutting kit. The RT-PCR product and vector recovered by enzyme digestion were connected at 16℃ for 16h under the action of T4 DNA ligase. The ligation product was transformed into Ecoli JM109 competent cells, coated with LB resistance plates containing 100mg / L ampicillin (Amp), and inverted at 37℃ for 16h. Single colonies were picked and shaken, plasmids were prepared with a plasmid extraction kit, double enzyme digestion and sequencing were performed, and the positive clones were recombinant engineering bacteria.

[0042] 4. Inducible expression of recombinant human TSA-1

[0043] A single colony of the recombinant engineered bacteria was picked and placed in LB culture medium containing 100 mg / L ampicillin, and cultured overnight at 250 rpm and 30°C. The overnight bacteria were expanded at a ratio of 1:100 to the early logarithmic growth stage, and IPTG (final concentration 1 mmol / L) was added. The expression of rhTSA-1 with a GST tag was induced at 250 rpm for 5 h. The bacterial precipitate was collected by centrifugation at 12000g and 4°C for 10 min, and resuspended in pH 7.3 PBS (containing 5 mmol / L DTT). The bacteria were disrupted by ultrasonication, and the supernatant was collected by centrifugation at 12000g and 4°C for 20 min. The target peak was purified and collected by Glutathione sepharose 4FastFlow column, and the expression of rhTSA-1 was analyzed by SDS-PAGE electrophoresis.

[0044] The results of the above experiment are as follows:

[0045] 1. Amplification results of TSA-1 cDNA

[0046] The human TSA-1 coding cDNA was obtained by RT-PCR, including the start codon, stop codon and restriction site sequence of nearly 500 bp (see Figure 1 ). Figure 1 The electrophoresis results of the RT-PCR amplification products of human TSA-1, lane 1: DNA molecular weight standard (2000, 1500, 750, 500, 250, 100 bp from top to bottom); lane 2: PCR product.

[0047] The nucleic acid molecule encoding the recombinant human testis-specific antigen is shown in SEQ ID NO: 2:

[0048] ATGAGAAAATGTTGTCCAGAGGTGGCAAGTGGTGTGTCCAAAGTAACACAGCAGGTAAGTGGCAGAGTCGGGGATTTGAATACCAAGTTTGTGATTCTAAGTGCAGAATTGAGAGATGCTTTTGACTCACATTGCGTCTTCCTGTCTGCTTGGCTGACCAGGTGGGTACGGAACCTTGAGGCACCTGGCTTGCTTCTAGGTAGGCAGAGTTGCTGTGTGATTGCCCAGCCAGGGGC CAGGCATTTTGAGTTGAGCAACTCCCTTGGCAGAAGGCCACATACCTGCCCATTTTCCATCAAAATGTCCCCAGTGCCACAGCACCCAAGCTTCCCTTCCCCCAACAGGTGGATGTTTTTGAAAAGAAACCACACAGAGATTTTGTTACTTTTTTCTTTTTTTTTTAAGTATTCATGAGAATTCTCCCTCTGCAGGGTTAAAATGCATGTTCTTGGGAAAAAGTGGTATTTAA.

[0049] 2. Identification results of pGEX / TSA-1 expression plasmid

[0050] The recombinant plasmid pGEX / TSA-1 was identified by double digestion with SalⅠ and SmaⅠ. The results showed that the insert fragment was obtained by double digestion. The cDNA fragment encoding TSA-1 had been cloned into the expression vector pGEX-4T-2. The structure of the recombinant expression plasmid pGEX / TSA-1 was correct ( Figure 2 ). The recombinant plasmid was analyzed by DNA sequence, and the cDNA sequence was consistent with the sequence reported in the literature. Figure 2 The results of enzyme digestion identification of the recombinant plasmid pGEX / TSA-1, where lane 1: pGEX-4T-2 plasmid digested with SalⅠ and SmaⅠ; lane 2: gene amplification product of human TSA-1; lane 3: pGEX / TSA-1 recombinant plasmid digested with SalⅠ and SmaⅠ; lane 4: DNA molecular weight standard (2000, 1500, 750, 500, 250, 100 bp from top to bottom).

[0051] 3. Inducible expression of recombinant human TSA-1

[0052] The induced pGEX / TSA-1 engineering bacteria and purified rhTSA-1 (recombinant human TSA-1) were subjected to SDS-PAGE electrophoresis analysis, and an obvious new protein band appeared at a molecular weight of about 13kD ( Figure 3 ), no obvious new bands were observed in the control. Figure 3 The induced expression results of rhTSA-1, wherein lane 1: induced expression of engineered bacteria; lane 2: impurity protein; lanes 3, 4, 5: target protein; lane 6: low molecular weight protein molecular weight standard (3.5, 10, 15, 20, 30, 40, 50, 60, 80, 110, 160, 260 kDa).

[0053] The amino acid sequence of the recombinant human testis-specific antigen is shown in SEQ ID NO: 1:

[0054] SEQ ID NO: 1:

[0055] MRKCCPEVASGVSKVTQQVSGRVGDLNTKFVILSAELRDAFDSHCVFLS AWLTRWVRNLEAPGLLLGRQSCCVIAQPGARHFELSNSLGRRPHTCPFSIKMS PVPQHPSFPSPNRWMFLKRNHTEILLLFLFFLSIHENSPSAGLKCMFLGKSGI.

[0056] Example 2

[0057] In this example, the prepared recombinant human testis-specific antigen was used to detect serum samples.

[0058] 1. Materials

[0059] Peripheral blood serum samples of 40 female patients with positive antisperm antibodies who visited the reproductive center of a tertiary hospital were collected, with an age range of 30-40 years old. The commercially available antisperm antibody detection kit (a kit for whole sperm antigen) and the recombinant human testis-specific antigen prepared in Example 1 were used for detection.

[0060] 2. The specific experimental steps are as follows

[0061] The ELISA plate was coated with the prepared recombinant human testis-specific antigen (rhTSA-1) at 10 ng / well, and the relevant solution was prepared according to the instructions of the anti-sperm antibody detection kit. After discarding the coating solution, the specimen diluent, the serum specimen to be tested and the control, the enzyme conjugate, the substrate and the stop solution were added in sequence. Each part was washed 5 times with washing solution in between steps. After color development, the OD value of each well was read at 450 nm with an ELISA reader.

[0062] 3. The test results are as follows

[0063] The results showed that the positive rate of TSA-1 antibodies in the patients' peripheral blood serum was significantly lower than the results of clinical anti-sperm antibody detection (Table 1).

[0064] Table 1

[0065]

[0066]

[0067] Thousands of sperm membrane proteins have been discovered so far, but not all sperm membrane antigens can induce the body to produce antibodies, and the induced antibodies combined with related antigens may not necessarily lead to infertility. Infertility-related ASA mainly affects normal fertility by affecting sperm capacitation, acrosome reaction, sperm-egg fusion and embryo implantation, while most ASA corresponding to sperm antigens do not cause clinical infertility. Therefore, the detection of ASA corresponding to sperm antigens related to infertility will play a precise and decisive role in the clinical diagnosis and treatment of infertility.

[0068] The detection antigen in the anti-sperm antibody detection kit currently used in clinical practice is the whole sperm antigen. Even if the test result is positive, the proportion of sperm antigens that cause immune infertility is very low, and the corresponding proportion of real immune infertility is very low (for patients who tested positive using the anti-sperm antibody detection kit, it was proved in subsequent clinical practice that they were not really immune infertility). Therefore, the positive results of ordinary anti-sperm antibody detection kits have little clinical reference value, and only negative results have a certain reference value. This is a consensus in clinical diagnosis and treatment and an urgent problem to be solved. Using infertility-related sperm proteins as detection antigens to accurately detect the corresponding ASA can solve this specificity problem well.

[0069] In summary, the present invention successfully cloned the coding gene of human TSA-1, induced the expression of recombinant human TSA-1, and established a corresponding purification process. The purity of the prepared recombinant human TSA-1 exceeded 98%, which laid a certain foundation for the diagnosis and treatment of immune infertility and the development of contraceptive vaccines.

[0070] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. Use of recombinant human testis-specific antigen in the preparation of infertility diagnostic drugs or contraceptive drugs, characterized in that: The amino acid sequence of the antigen includes that shown in SEQ ID NO:

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the recombinant human testis-specific antigen according to claim 1; Preferably, the nucleotide sequence of the nucleic acid molecule includes that shown in SEQ ID NO:

2.

3. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 2; Preferably, the expression vector is obtained by connecting the nucleic acid molecule according to claim 2 to the pGEX-4T-2 vector.

4. A host cell, characterized in that The host cell contains at least one copy of the expression vector according to claim 3; or the genome of the host cell is integrated with the nucleic acid molecule according to claim 3; Preferably, the host cell is Escherichia coli.

5. A method for preparing a recombinant human testis-specific antigen, characterized in that: The preparation method comprises: (1) RNA was extracted from human testicular tissue by reverse transcription PCR amplification to obtain a gene encoding a human testicular specific antigen, wherein the upstream primer sequence of the reverse transcription PCR amplification is shown in SEQ ID NO: 3; and the downstream primer sequence of the amplification is shown in SEQ ID NO: 4; (2) constructing the obtained coding gene on the expression vector pGEX-4T-2 and transferring it into a competent host bacterium to obtain a recombinant expression engineering bacterium; (3) culturing the recombinant expression engineering bacteria, inducing expression, and then purifying to obtain the recombinant human testis-specific antigen.

6. The method for preparing the recombinant human testis-specific antigen according to claim 5, characterized in that: The steps of constructing the coding gene include: double-digesting the obtained coding gene fragment and the expression vector pGEX-4T-2 with restriction endonucleases respectively, purifying the obtained coding gene fragment and the open-circle expression vector after digestion, and connecting the coding gene fragment and the open-circle expression vector to obtain a recombinant expression vector.

7. The method for preparing the recombinant human testis-specific antigen according to claim 6, characterized in that: The restriction endonucleases include SmaI and SalI.

8. The method for preparing a recombinant human testis-specific antigen according to any one of claims 5 to 7, characterized in that: The host bacteria is selected from: Escherichia coli TG1 or Escherichia coli Ecoli JM109.

9. The method for preparing a recombinant human testis-specific antigen according to any one of claims 5 to 8, characterized in that: The transfer method includes: chemical transfection or electroporation.

10. Use of the nucleic acid molecule according to claim 2, the expression vector according to claim 3, the host cell according to claim 4 or the method for preparing the recombinant human testis-specific antigen according to any one of claims 5 to 9 in the preparation of infertility diagnostic drugs or contraceptive drugs.

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