C3 protein, a non-syndromic prenatal non-invasive diagnostic molecular marker for cleft lip and palate, and its application.

Abstract

The present invention discloses a protein molecular marker C3 for non-invasive prenatal diagnosis of non-syndromic cleft lip and palate fetuses and its application, belonging to the field of pharmaceutical biotechnology. The present invention utilizes proteomics detection platform technology and ELISA experimental methods to compare differentially expressed proteins in the serum of pregnant women with non-syndromic cleft lip and palate fetuses and pregnant women with healthy fetuses, and then analyzes the correlation between the differentially expressed proteins and the disease. Based on a large number of specimens from the training cohort and the validation cohort, it was discovered and confirmed for the first time that abnormal expression of protein C3 in the serum of pregnant women is closely correlated with the occurrence of non-syndromic cleft lip and palate fetuses, and can be used alone for prenatal screening of non-syndromic cleft lip and palate, providing a new target for the treatment of non-syndromic cleft lip and palate, which has great clinical significance.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical biotechnology, and in particular relates to a protein molecule marker C3 for non-invasive prenatal diagnosis of non-syndromic cleft lip and palate fetuses and an application thereof. Background Art

[0002] Researching non-invasive early embryonic diagnosis methods for non-syndromic cleft lip and palate will enable diagnosis before serious structural abnormalities or irreversible damage occur, and develop corresponding new strategies for early embryonic treatment and prevention, which are extremely important for reducing the disability rate of deformities and improving the quality of the population.

[0003] Establishing a non-invasive early screening method for congenital malformations has always been an ideal goal that people have been pursuing. Although the current imaging technology (ultrasound and MRI) has developed very rapidly, which has advanced the diagnosis time of some congenital malformations, it still cannot meet the requirements of early screening and diagnosis. In addition, the maxillary alveolar process blocks the ultrasound beam, making it difficult to reach the cleft palate, so the detection rate of single cleft palate is extremely low. Therefore, it is particularly important to find early non-invasive prenatal diagnostic molecular markers, which also provides a basis for early consultation and treatment. Maternal serological examination is a non-invasive prenatal diagnostic method that is easy for pregnant women to accept and suitable for large-scale prenatal screening. Therefore, many scholars at home and abroad are committed to the research of discovering new diagnostic specific markers, but so far, except for neural tube defects and Down syndrome, which can be screened by serum alpha-fetoprotein, there are no diagnostic molecular markers for other birth defects that have been clinically applied.

[0004] In recent years, with the rapid development of various omics technologies, a series of new technologies have been integrated into high-throughput omics research, which has made new breakthroughs in the screening of disease diagnostic molecular markers and provided an extremely important means for the translational medicine research that everyone attaches great importance to. Proteomics is a very promising high-throughput omics research method for studying pathological changes and screening disease diagnostic biomarkers. The study of serum proteomics in pregnant women can fully consider both maternal and fetal factors, screen out one or a group of key molecules with obvious changes from many complex proteins, and comprehensively analyze their changing patterns, which will be conducive to determining molecular markers for early diagnosis of diseases and prognosis determination, and is also the trend of converting complex molecular markers into clinical applications in the future. Summary of the invention

[0005] In response to the above problems, the purpose of the present invention is to provide a molecular marker for non-invasive prenatal diagnosis of non-syndromic cleft lip and palate in fetuses and its application. The molecular marker is C3 protein, which can be used alone for prenatal screening of non-syndromic cleft lip and palate, providing a new target for the treatment of non-syndromic cleft lip and palate.

[0006] In order to achieve the above object, the present invention provides the following technical solutions.

[0007] The present invention provides the use of C3 protein in preparing a product for non-invasive prenatal diagnosis and screening of non-syndromic cleft lip and palate fetuses.

[0008] Furthermore, the non-invasive prenatal diagnostic screening product for non-syndromic cleft lip and palate fetuses uses C3 protein as a diagnostic marker.

[0009] Furthermore, the non-invasive prenatal diagnostic screening product for non-syndromic cleft lip and palate fetuses detects the expression level of C3 protein in pregnant women's blood, urine, exosomes, amniotic fluid and malformed fetuses or children's specimens through chips (protein chips and microfluidic chips, etc.), digital single-molecule immune arrays, ELISA, radioimmunoassay, immunoturbidimetry, immunohistochemistry, and Western blotting. The degree of C3 protein expression is negatively correlated with the incidence of non-syndromic cleft lip and palate fetuses.

[0010] Furthermore, the product includes reagents, kits, iTRAQ labels, chromatographic fractionation, and liquid chromatography-tandem mass spectrometry (LC-MS / MS) data acquisition technology to detect C3 protein in samples.

[0011] Furthermore, the test samples include blood, urine, exosomes, amniotic fluid and malformed fetuses or sick children.

[0012] The present invention also provides a method for preparing a non-invasive prenatal diagnosis tool for a fetus with non-syndromic cleft lip and palate using a reagent for detecting molecular markers for non-invasive prenatal diagnosis of a fetus.

[0013] Furthermore, the reagents for detecting molecular markers for non-invasive prenatal diagnosis of non-syndromic cleft lip and palate fetuses include reagents capable of quantifying C3 protein.

[0014] Furthermore, the tools for prenatal screening, early warning and diagnosis of non-syndromic cleft lip and palate fetuses include antibodies, protein chips, ELISA kits, high-throughput sequencing platforms and Western blotting, etc.

[0015] The reagents for quantitative protein of the present invention include antibodies or fragments thereof that specifically bind to proteins. Antibodies or fragments thereof of any structure, size, immunoglobulin class, origin, etc. can be used as long as it binds to the target protein. The antibodies or fragments thereof included in the detection product of the present invention can be monoclonal or polyclonal. Antibody fragments refer to a portion of an antibody that retains the binding activity of the antibody to the antigen or a peptide containing a portion of an antibody. The reagents for quantitative protein of the present invention can include an isolated nucleic acid encoding an amino acid sequence of an antibody or an antibody fragment, a vector containing the nucleic acid, and a cell carrying the vector.

[0016] Compared with the prior art, the present invention has the following beneficial effects.

[0017] The present invention discovers and confirms for the first time that abnormal expression of protein C3 in pregnant women's serum is closely related to the occurrence of non-syndromic cleft lip and palate in fetuses, with a large number of samples verified and accurate results.

[0018] The present invention provides protein markers related to non-invasive prenatal diagnosis of non-syndromic cleft lip and palate fetuses, provides services for prenatal diagnosis or risk monitoring of non-syndromic cleft lip and palate fetuses, and sells diagnostic and prognostic consulting services to hospitals and clinics in a collaborative or independent manner.

[0019] The protein markers related to the non-invasive prenatal diagnosis of non-syndromic cleft lip and palate fetuses provided by the present invention provide a new approach for prenatal screening, early warning and diagnosis of non-syndromic cleft lip and palate fetuses. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The differentially expressed protein profiles screened by proteomics. (A) Cluster analysis heat map of differentially expressed proteins between the non-syndromic cleft lip and palate group and the control group; (B) Volcano map of differentially expressed proteins between the non-syndromic cleft lip and palate group and the control group.

[0021] Figure 2 . GO and KEGG functional enrichment analysis of differentially expressed proteins. Among them, (A) GO functional enrichment analysis of differentially expressed proteins. (B) KEGG functional enrichment analysis of differentially expressed proteins.

[0022] Figure 3 . Expression level and diagnostic efficacy of protein C3. (A) Protein expression level of C3 in the control group and non-syndromic cleft lip and palate group. (B) ROC curve analysis result of C3. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The maternal peripheral blood specimens used were derived from the Shengjing Birth Cohort Sample Library and were approved by the Ethics Committee of Shengjing Hospital Affiliated to China Medical University (Approval No.: 2017PS264K).

[0024] Example 1: iTRAQ quantitative proteomics technology was used to screen protein diagnostic molecular markers in the training cohort.

[0025] 1. Separate serum.

[0026] Collect whole blood samples in a coagulant tube, mix gently by inversion, and centrifuge at 1600×g for 10 min in a 4°C low-temperature centrifuge. Collect the supernatant (serum) into a new EP tube after centrifugation at 16000×g for 10 min to remove cell debris. Divide the serum into multiple centrifuge tubes and freeze them in a -80°C low-temperature refrigerator.

[0027] 2. iTRAQ quantitative proteomics detection process.

[0028] Serum from 30 pregnant women with healthy fetuses and 30 pregnant women with non-syndromic cleft lip and palate fetuses was collected. The clinical characteristics are shown in Table 1. Ten samples in each group were mixed into one tube, that is, each group contained three biological replicates, a total of six samples. The detection process included protein extraction, peptide enzymatic hydrolysis, iTRAQ labeling, chromatographic fractionation, liquid chromatography-tandem mass spectrometry (LC-MS / MS) data acquisition, protein identification and quantitative analysis.

[0029] Table 1. Clinical characteristics of pregnant women used for proteomic testing

[0030] .

[0031] 3. Differential protein profiles screened by proteomics.

[0032] A total of 1055 proteins were identified, and differentially expressed proteins were screened according to the criteria of differential expression fold change of more than 1.2 times (upregulation greater than 1.2 times or downregulation less than 0.83 times) and p<0.05. This project used a hierarchical clustering algorithm to perform cluster analysis on differentially expressed proteins and displayed the data in the form of a heat map. Compared with the control group, there were 39 upregulated differentially expressed genes and 43 downregulated differentially expressed genes in the non-syndromic cleft lip and palate group ( Figure 1 ).

[0033] 4. Functional enrichment analysis of differentially expressed proteins.

[0034] Blast2Go software was used to perform GO functional annotation on all proteins identified in this project, and then GO functional enrichment analysis of differentially expressed proteins was performed using Fisher's exact test method, such as Figure 2As shown in A, the analysis results showed that in the non-syndromic cleft lip and palate group, important biological processes such as positive regulation of alpha-beta T cell activation, regulation of alpha-beta T cell activation, alpha-beta T cell activation, negative regulation of immune effector process, positive regulation of fibrinolysis, molecular functions such as phospholipase inhibitor activity, lipase inhibitor activity, protein phosphorylated amino acid binding, phosphoserine residue binding, beta-N-acetylglucosaminylglycopeptidebeta-1,4-galactosyltransferase activity, and localized proteins such as PML body, Golgi cisterna, Golgistack, extrinsic component of external side of plasma membrane, mitochondrial protein complex, had significant changes. KEGG pathway enrichment analysis of differentially expressed proteins was performed using the Fisher exact test method. Figure 2 B The results showed that the important pathways of Pentose phosphate pathway and Mannose type O-glycan biosyntheis had significant changes.

[0035] Example 2 validates the diagnostic efficacy of protein C3 as a molecular marker in a validation cohort.

[0036] 1. Include the research sample.

[0037] A total of 43 pregnant women with healthy fetuses and 43 pregnant women with non-syndromic cleft lip and palate fetuses were included in the validation cohort. Serum was separated according to the method in Example 1.1.

[0038] 2. ELISA was used to detect the expression of protein C3.

[0039] Take 50µL of serum and dilute it to 100µL in PBS liquid, then use the human C3 ELISA detection kit, follow the steps in the instructions, add the reaction stop solution, and use a multifunctional microplate reader to detect at 450nm, and calculate the C3 expression level according to the standard curve. The test results show that compared with the normal pregnant women group, the expression level of C3 in the serum of non-syndromic cleft lip and palate fetuses is significantly lower, and there is a statistical difference ( Figure 3 A).

[0040] 3. Draw the ROC curve.

[0041] ROC curve analysis showed that the sensitivity of C3 in diagnosing non-syndromic cleft lip and palate was 76.7% and the specificity was 83.7%. The area under the ROC curve was 0.84, p<0.001. The results showed that C3 can be used as a molecular marker for prenatal diagnosis of non-syndromic cleft lip and palate fetuses ( Figure 3 B).

[0042] In summary, the present invention utilizes the proteomics detection platform technology to extract proteins and perform peptide fragmentation enzymatic hydrolysis on the serum biological samples of the subjects, followed by iTRAQ labeling and SCX chromatographic fractionation. Each fractionated sample is separated using the HPLC liquid phase system Easy nLC with a nanoliter flow rate. The software Mascot2.2 and Proteome Discoverer1.4 are used for library identification and quantitative analysis. By comparing the differences in protein expression between disease patients and normal people, it is analyzed which protein expression abnormality is related to the disease. The samples were further expanded, and the potential diagnostic protein molecular markers obtained by proteomics were verified by ELISA, and a universal non-syndromic cleft lip and palate risk warning model was established using the ROC curve. The results showed that C3 can be used as a molecular marker for prenatal diagnosis of non-syndromic cleft lip and palate fetuses, providing a new target for the treatment of non-syndromic cleft lip and palate.

Claims

1. Application of C3 protein in the preparation of a product for non-invasive prenatal diagnosis and screening of non-syndromic cleft lip and palate fetuses, wherein the product for non-invasive prenatal diagnosis and screening of non-syndromic cleft lip and palate fetuses uses C3 protein as a diagnostic marker.

2. The use according to claim 1, characterized in that: The product for non-syndromic cleft lip and palate fetal prenatal non-invasive diagnosis and screening detects the expression level of C3 protein in the sample through ELISA, radioimmunoassay, immunoturbidimetry, immunohistochemistry, and Western blotting. The degree of C3 protein expression is negatively correlated with the incidence of non-syndromic cleft lip and palate fetuses.

3. Use of a reagent for detecting a molecular marker for non-invasive prenatal diagnosis of non-syndromic cleft lip and palate in the preparation of a non-invasive prenatal diagnosis tool for the fetus, wherein the molecular marker is C3 protein, and the reagent includes a reagent capable of quantitatively detecting C3 protein.

4. The use according to claim 3, characterized in that: The non-invasive diagnostic tools include protein chips, ELISA kits, high-throughput sequencing platforms and Western blotting.

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