阅读说明：本技术 一种杂交瘤细胞株、抗旋毛虫新生幼虫期丝氨酸蛋白酶的单克隆抗体及应用 (hybridoma cell strains, monoclonal antibody of trichina-resistant serine protease in new larva stage and application ) 是由 刘明远 刘晓雷 杨勇 P·布瓦罗 I·瓦利 于 2019-09-30 设计创作，主要内容包括：一种杂交瘤细胞株、抗旋毛虫新生幼虫期丝氨酸蛋白酶的单克隆抗体及应用,属于旋毛虫(T.spiralis)的防治领域。针对如何对旋毛虫病进行特异性诊断的技术问题,本发明提供了一种杂交瘤细胞株,其保藏编号为CGMCC No.18319以及由上述杂交瘤细胞株所分泌单克隆抗体。所述单克隆抗体能够特异性的识别ASKEDS氨基酸基序。本发明建立旋毛虫(T.spiralis)血清学诊断方法奠定了基础。(hybridoma cell strains, monoclonal antibodies of trichina-resistant serine protease in the new larva period and application, belong to the field of prevention and treatment of trichina (T.spiralis), aiming at the technical problem of how to carry out specificity diagnosis on trichina, the invention provides hybridoma cell strains, the preservation number of which is CGMCC No.18319, and the monoclonal antibodies secreted by the hybridoma cell strains.)

1, hybridoma cell strains with the preservation number of CGMCC No. 18319.

monoclonal antibodies against Trichinella spiralis neonatal larval serine protease secreted by the hybridoma cell line of claim 1.

3. The monoclonal antibody according to claim 2, wherein the epitope recognized by the monoclonal antibody is represented by SEQ ID No. 1.

4. Use of the hybridoma cell line of claim 1 for the preparation of a reagent for diagnosing t. spiralis infection of trichina.

Application of the epitope polypeptide shown in SEQ ID NO.1 in preparation of vaccines for diagnosing or preventing trichina T.

Technical Field

The invention belongs to the field of prevention and treatment of trichina (T.spiralis), and particularly relates to hybridoma cell strains, a monoclonal antibody for resisting serine protease in a new larva stage of trichina and application.

Background

Trichinemia is mainly caused by the uncooked or semi-uncooked meat containing trichinemia of the host. The incubation period of the human trichinosis is 2 weeks on average, the main symptoms of the acute patient are fever, severe muscle pain, severe diarrhea, facial edema, eosinophilia and the like, the symptoms can last for several weeks and cause body failure, and particularly, severe infected patients can have severe damage to cardiac muscle and brain and even die.

In view of the great threat and harm of trichina to public health safety and human health, trichina is classified as a mandatory disease species for slaughter animal mandatory inspection and first inspection by the world animal health Organization (OIE). in China, the trichina is directly classified as a serious disease species, and is which is a typical disease species capable of causing sudden public health events except for strong zoonosis.

Researchers at home and abroad have conducted a great deal of research on trichina detection methods, such as indirect fluorescence immunoassay, immunoenzyme staining assay, immunoblotting assay, immunoadsorption assay (ELISA), etc., and the enzyme-linked immunosorbent assay (ELISA) is the most common immunological method for trichina infection detection, which has high sensitivity and can detect 1 larva per 100g of muscle tissue, at present, the ES antigen of trichina muscle larva excretion is the standard antigen for serological antibody detection of OIE and only specified by the international trichina committee.

At present, a trichina newborn larva differential cDNA library is successfully constructed by utilizing a differential subtraction hybridization SSH (hybridization), trichina newborn larva high-reactogenicity and high-specificity antigen genes, namely serine protease genes, are firstly screened and cloned, and the genes are named as Ts-T668.Ts-T668 and consist of enzyme active regions and C terminal regions, but the functions of the C terminal regions are unclear.

Disclosure of Invention

Aiming at the technical problem of how to carry out specific diagnosis on trichinosis, the invention provides hybridoma cell strains, the preservation number of which is CGMCC No. 18319.

The invention also provides monoclonal antibodies of the trichina resisting serine protease in the new larva stage, which are secreted by the hybridoma cell strain CGMCC No. 18319.

, the amino acid sequence of the epitope polypeptide recognized by the monoclonal antibody is shown in SEQ ID No. 1.

The hybridoma cell strain can be used for preparing a reagent for diagnosing T.spiralis infection, and the epitope polypeptide shown by SEQ ID NO.1 can be used for preparing a vaccine for diagnosing or preventing T.spiralis infection.

Advantageous effects

The monoclonal antibody has the advantages of strong specificity combined with antigen, good purity, strong repeatability, convenient quality control, good affinity and mass production, and is widely applied to the establishment of an immunological detection method by , so that hybridoma cell strains which can secrete anti-Ts-T668-C protein specific antibodies are screened, and Ts-T668-C protein specific B cell epitopes recognized by the secreted monoclonal antibodies are identified, so that the improvement of a diagnostic method for trichinosis and the development of subunit vaccines are of great significance.

The monoclonal antibody prepared by the invention specifically reacts with the antigen of the newborn larva of trichina and the crude extract of the 6-day-old adult. Immunofluorescence shows that the obtained monoclonal antibody specifically recognizes the embryo of the newborn larva and the adult worm with the age of 6 days, and does not react with the worm bodies in other periods. The test result of Ts-T668-C competition ELISA shows that the monoclonal antibody Ts-T668-5D4-Ab can compete with trichina infection positive pig serum to bind with Ts-T668-C antigen.

The invention utilizes peptide scanning technology to identify and determine the B cell epitope of the Ts-T668-C antigen recognized by the Ts-T668-5D4-AbThe Ts-T668-5D4-Ab recognition peptide is³²⁷NSPEGTVKWASKEDS³⁴¹And³³⁶ASKEDSPVDLSTASR³⁵⁰it contains 6 common amino acid motifs, "ASKEDS," which are the antigen-recognizing epitopes of this monoclonal antibody.

Drawings

FIG. 1 shows SDS-PAGE results of purified Ts-T668-C protein, M is protein molecular weight standard; 1. rTs-T668-C protein after purification;

FIG. 2 shows the result of detecting the competitive binding of monoclonal antibody 5D4 and positive serum of trichina suis infection with Ts-T668-C protein, the ordinate is positive quality control serum OD_450nmAbsorbance (P) and negative quality control serum OD_450nmThe specific value of the absorbance (N) and the abscissa is the dilution multiple of the quality control serum;

FIG. 3 is a graph showing the specific recognition result of the monoclonal antibody 5D4 on rT668-C protein, M: protein marker.1.SP2/0 cell culture solution, 2. hybridoma cell culture solution supernatant, 3. mice infected with 300 trichina positive serum;

FIG. 4 is a graph showing the recognition results of the monoclonal antibody on the trichina bodies in different developmental stages, N: SP2/0 cell culture solution, rT668 anti-T668-C monoclonal antibody, wherein 1 is crude extract of muscle larva, 2 is crude extract of adult of 6 days old, and 3 is crude extract of newborn larva;

FIG. 5 is a graph of immunofluorescence analysis results of monoclonal antibodies recognizing different stages of trichina development, wherein N: SP2/0 cell culture fluid, rT668-C, anti-T668-C monoclonal antibody; AD: 6-day-old adults, NBL: newborn larvae, ML: muscle larvae;

FIG. 6 indirect ELISA assay results, with different groups (different synthetic peptide coated antigens) on the abscissa and OD450nm absorbance on the ordinate.

Detailed Description

The method comprises the steps of carrying out immunological screening on cDNA libraries of trichina neonatal larvae to obtain antigen proteins with high abundance and strong reactogenicity, wherein the antigen proteins are named as T668, and the T668 protein belongs to serine protease through bioinformatics sequence analysis.

The invention adopts TRIZOL to extract trichina (T.spiralis) total RNA, and then utilizes a reverse transcription technology to clone Ts-T668-C (C end immunodominance region), inserts the cDNA into a prokaryotic expression vector pET28a, utilizes pET28a prokaryotic expression vector to carry out prokaryotic expression on Ts-T668-C gene, immunizes BALB/C mice with the expressed soluble Ts-T668-C through nickel ion metal affinity chromatography purification, takes spleen cells thereof to fuse with SP2/0 myeloma cells to prepare hybridoma cells, and uses the recombined Ts-T668-C as an antigen for detection to establish an indirect ELISA detection method to screen the positive hybridoma cells, finally obtains hybridoma cell strains which stably secrete anti-Ts-T-C protein monoclonal antibodies, and the hybridoma cell strains are preserved in China general microbiological culture collection center in 8-15 days in 2019 and named as CGMCC No.18319, and the application is NBL-668D 385D 4.

The main experimental materials and sources used in the present invention are as follows:

1. main reagents and drugs: ni purification column histrap ph was purchased from GE corporation, usa; fetal bovine serum, 1640 medium was purchased from Biological Industries; HAT medium (50X), HT medium (50X) and antibody subclass identification kit were purchased from sigma; a solvent TMB substrate Solution was obtained from TIANGEN; horseradish peroxidase (HRP) labeled goat anti-mouse IgG antibodies were purchased from beijing boolsen; the prestained protein Marker was purchased from Fermentas; restriction enzymes EcoRI and XhoI, reverse transcriptase, Ex Taq DNA polymerase, T4DNA ligase were purchased from Takara Bio (Dalian) Ltd; ECL luminogenic substrates were purchased from beijing solibao corporation.

2. Experimental animals: 6 week old BALB/c mice were provided by Vinca hundred million laboratory animal technology, Inc.

The technical scheme of the invention is described in detail below, and the molecular biological experimental methods such as reverse transcription, PCR, enzyme digestion and the like in the following examples are all conventional technical methods in the field unless otherwise specified.