阅读说明：本技术 一种特异性识别蛋白drc1的多克隆抗体的制备方法及其应用 (A kind of preparation method and applications of the polyclonal antibody of specific recognition protein D RC1 ) 是由 刘明兮 刘思钰 吴佳艳 于 2019-07-26 设计创作，主要内容包括：本发明涉及生物科学和检测试剂技术领域,具体公开了一种特异性识别蛋白DRC1的多克隆抗体的制备方法,包括如下步骤：(1)蛋白DRC1抗原表位分析：通过抗原表位分析选择目的片段用作蛋白DRC1的重组表达,所述目的片段的基因序列如序列表中SEQ ID N0.1所示；(2)将选定的目的片段的DNA通过PCR扩增后插入到表达载体构建含有如SEQ ID NO.1所示核昔酸序列的重组表达载体,将所述重组表达载体转化大肠杆菌感受态细胞获得重组蛋白DRC1抗原；(3)将所述重组蛋白DRC1抗原免疫动物,经血清获得特异性识别蛋白DRC1的多克隆抗体。本发明在重组蛋白制备的中采用大肠杆菌表达系统,能很好的保证重组蛋白的表达,表达产量高,有利于大批量的制备特异性识别蛋白DRC1的多克隆抗体。(The present invention relates to bioscience and detection reagent technical field, specifically disclose a kind of preparation method of the polyclonal antibody of specific recognition protein D RC1, include the following steps: (1) protein D RC1 Characterization of antigenic epitopes: selecting target fragment to be used as the recombinant expression of protein D RC1 by Characterization of antigenic epitopes, the gene order of the target fragment is as shown in SEQ ID N0.1 in sequence table；(2) recombinant expression carrier conversion competent escherichia coli cell is obtained recombinant protein DRC1 antigen by the recombinant expression carrier that the DNA of selected target fragment is contained the core former times acid sequence as shown in SEQ ID NO.1 by being inserted into expression vector establishment after PCR amplification；(3) by the recombinant protein DRC1 antigen-immunized animal, the polyclonal antibody of specific recognition protein D RC1 is obtained through serum.The present invention prepares middle using escherichia expression system in recombinant protein, can guarantee the expression of recombinant protein well, expression yield is high, is conducive to the polyclonal antibody of large batch of preparation specific recognition protein D RC1.)

1. a kind of preparation method of the polyclonal antibody of specific recognition protein D RC1, which comprises the steps of:

(1) protein D RC1 Characterization of antigenic epitopes: the recombination table for selecting target fragment to be used as protein D RC1 by Characterization of antigenic epitopes It reaches, the gene order of the target fragment is as shown in SEQ ID N0.1 in sequence table；

(2) DNA of selected target fragment is contained by being inserted into expression vector establishment after PCR amplification such as SEQ ID NO.1 The recombinant expression carrier of shown core former times acid sequence is recombinated recombinant expression carrier conversion competent escherichia coli cell Protein D RC1 antigen；

(3) by the recombinant protein DRC1 antigen-immunized animal, the Anti-TNF-α of specific recognition protein D RC1 is obtained through serum Body.

2. the preparation method of the polyclonal antibody of specific recognition protein D RC1 according to claim 1, which is characterized in that The primer sequence of PCR amplification is shown in SEQ ID No.2 and SEQ ID No.3 in the step (2).

3. the preparation method of the polyclonal antibody of specific recognition protein D RC1 according to claim 2, which is characterized in that Expression vector in the step (2) is PET28a.

4. the preparation method of the polyclonal antibody of specific recognition protein D RC1 according to claim 3, which is characterized in that The E. coli competent is e. coli bl21 (DE3) competent cell.

5. the preparation method of the polyclonal antibody of specific recognition protein D RC1 according to claim 4, which is characterized in that The immune animal of recombinant protein DRC1 antigen is new zealand white rabbit, ICR mouse in the step (3).

6. a kind of preparation method of the polyclonal antibody of any one of claim 1-5 specific recognition protein D RC1 is being tested Application in sample in the quantitative detection of protein D RC1.

Technical field

The present invention relates to bioscience and detection reagent technical field.

Background technique

DRC1 albumen (dynein adjust compound protein 1, Dynein regulatory complex protein 1) by DRC1 gene expression.DRC1 participates in coding connection albumen dynein and adjusts compound (nexin-dynein regulatory Complex, N-DRC) center compositions, adjust the assembling of ciliary dynein.DRC1 and DRC2 is assembled jointly to be formed in N-DRC Cardiac skeleton and its connection with external two-tube micro-pipe.The mutation of the gene can cause the dyskinesia of cilium.Cilium is shown in research Abnormal motion and various respiratory tract disease are closely related, such as nasosinusitis, COPD, cystic fibrosis (cystic Fibrosis), primary ciliary dyskinesia (PCD, Primary ciliary dyskinesia) etc. (bibliography: Jing, J.C.and J.J.Chen,et al.(2017)."Visualization and Detection of Ciliary Beating Pattern and Frequency in the Upper Airway using Phase Resolved Doppler Optical Coherence Tomography."Sci Rep 7(1):8522.)。

Clinically multiple exhibition is chronic respiratory tract infection to the relevant disease of cilium.Researches show that the mutation of DRC1 and PCD Closely related (the bibliography: 1, Morimoto, K.and M.Hijikata, et al. (2019) " Recurring of morbidity large deletion in DRC1(CCDC164)identified as causing primary ciliary dyskinesia in two Asian patients."Mol Genet Genomic Med:e838.2、Raidt,J.and J.Wallmeier,et al.(2014)."Ciliary beat pattern and frequency in genetic variants of primary ciliary dyskinesia."Eur Respir J 44(6):1579-88.)。

At present respiratory disease has been not limited to the research of DRC1, because cilium in human body throughout extensive, grind both at home and abroad Study carefully personnel and probed between the expression degree and a variety of diseases of DRC1 and contacted, find the unconventionality expression of DRC1 except respiratory system, There are statistical correlations with disease with immune system, nervous system, reproductive system etc..

However the correlative study about protein D RC1 preparation method of polyclonal antibody is in blank rank in the prior art Section.

Summary of the invention

The purpose of the present invention is to provide a kind of preparation methods of the polyclonal antibody of specific recognition protein D RC1, with reality The preparation of the polyclonal antibody of existing specific recognition protein D RC1.

In order to achieve the above object, base case of the invention provides the Anti-TNF-α of specific recognition protein D RC1 a kind of The preparation method of body, includes the following steps:

(1) protein D RC1 Characterization of antigenic epitopes: the weight for selecting target fragment to be used as protein D RC1 by Characterization of antigenic epitopes Group expression, the gene order of the target fragment is as shown in SEQ ID N0.1 in sequence table；

(2) DNA of selected target fragment is contained by being inserted into expression vector establishment after PCR amplification such as SEQ ID The recombinant expression carrier of the acid sequence of core former times shown in NO.1 obtains recombinant expression carrier conversion competent escherichia coli cell Recombinant protein DRC1 antigen；

(3) by the recombinant protein DRC1 antigen-immunized animal, more grams of specific recognition protein D RC1 are obtained through serum Grand antibody.

Further, the primer sequence of PCR amplification is shown in SEQ ID No.2 and SEQ ID No.3 in step (2).

Further, the expression vector in step (2) is PET28a.

Further, E. coli competent is e. coli bl21 (DE3) competent cell.

Further, the immune animal of recombinant protein DRC1 antigen is new zealand white rabbit, ICR mouse in step (3).

The preparation method that base case of the invention provides the polyclonal antibody of specific recognition protein D RC1 a kind of is being tried Test the application in sample in the quantitative detection of protein D RC1.

Compared with prior art, the beneficial effects of the present invention are:

(1) preparation method of the polyclonal antibody of specific recognition protein D RC1 provided by the invention, passes through epitope Analysis selects suitable primer to be expanded the genetic fragment that can produce more strongly immunogenic site, and using the site Amino acid sequence preparation and reorganization protein fragments are immunized animal with this segment and obtain the antibody that specificity is directed to the site.It is recombinating The middle of albumen preparation uses escherichia expression system, can guarantee the expression of recombinant protein well, and expression yield is high, is conducive to The polyclonal antibody of large batch of preparation specific recognition protein D RC1.

(2) polyclonal antibody of specific recognition protein D RC1 prepared by the present invention can be applied to quantitative detection test specimen The residual level of middle protein D RC1, while being also convenient for albumen DRC1 under protein D RC1 purification media manufacturer monitoring specified conditions Fall off feature.

(3) polyclonal antibody of specific recognition DRC1 albumen of the invention is immune new zealand white rabbit and ICR mouse It obtains, both are all the common experimental animals for meeting laboratory practices, are easily obtained, and strain is stablized, and take its blood, are obtained Serum (serum need not purify) obtains antibody, and step is simple, meets modem animal ethical institution.ICR mouse is adaptable, body Lattice are healthy and strong, and reproductive capacity is strong, and the speed of growth is fast, and antibody specificity is high, and experimental repeatability is preferable.

Detailed description of the invention

Fig. 1 is the gradient glue Coomassie brilliant blue coloration result schematic diagram of protein D RC1 in the embodiment of the present invention 1；

Fig. 2 is the DRC1 hydrophily in embodiment 1, immunogenicity and epitope exposed property schematic diagram.

Specific embodiment

Below by the further details of explanation of specific embodiment: