阅读说明：本技术 非解乳糖链球菌表面粘附蛋白EF-Tu基因、氨基酸序列、原核表达蛋白和多克隆抗体 (Non-lactococcus lactis surface adhesion protein EF-Tu gene, amino acid sequence, prokaryotic expression protein and polyclonal antibody ) 是由 张景艳 张凯 王磊 张康 李建喜 王学智 仇正英 王贵波 辛蕊华 张丽娟 张宏 于 2020-03-09 设计创作，主要内容包括：本发明提供非解乳糖链球菌表面粘附蛋白EF-Tu基因序列,所述非解乳糖链球菌表面粘附蛋白EF-Tu基因序列为序列表中SEQ ID No.1。本发明还提供相应的氨基酸序列、原核表达蛋白、多克隆抗体及多克隆抗体的制备方法。利用本发明所提供的方法制备的多克隆抗体,能够特异性识别FGM菌EF-Tu蛋白,而不能识别FGM菌其他蛋白,在EF-Tu蛋白的检测和功能研究、菌株鉴别及乳酸菌益生性机制研究中具有广泛用途。(The invention provides an EF-Tu gene sequence of a non-lactococcus lactis surface adhesion protein, which is SEQ ID No.1 in a sequence table. The invention also provides a corresponding amino acid sequence, a prokaryotic expression protein, a polyclonal antibody and a preparation method of the polyclonal antibody. The polyclonal antibody prepared by the method provided by the invention can specifically recognize the EF-Tu protein of the FGM bacteria, but cannot recognize other proteins of the FGM bacteria, and has wide application in detection and function research of the EF-Tu protein, strain identification and probiotic mechanism research of lactic acid bacteria.)

1. The EF-Tu gene sequence of the non-lactococcus lactis surface adhesion protein is characterized in that: the gene sequence of the non-lactococcus lactis surface adhesion protein EF-Tu is SEQ ID No.1 in the sequence table.

2. The amino acid sequence of the non-lactococcus lactis surface adhesion protein EF-Tu protein is characterized in that: the amino acid sequence is SEQ ID No.2 in the sequence table.

3. The EF-Tu prokaryotic expression protein of the non-lactococcus lactis surface adhesion protein is characterized in that: constructing a recombinant expression vector containing a nucleotide sequence shown in SEQ ID No.3 in a sequence table, and transforming escherichia coli competent cells into the recombinant expression vector to obtain prokaryotic expression protein.

4. The preparation method of the non-lactococcus lactis surface adhesion protein EF-Tu protein polyclonal antibody is characterized by comprising the following steps of: the method comprises the following steps:

(1) constructing a recombinant expression vector containing a nucleotide sequence shown in SEQ ID No.3 in a sequence table, and transforming escherichia coli competent cells into the recombinant expression vector to obtain a recombinant protein antigen;

(2) immunizing animals with the recombinant protein antigen, and separating and purifying the serum to obtain the EF-Tu protein polyclonal antibody of the non-lactococcus lactis surface adhesion protein.

5. The method of claim 4, wherein: the recombinant expression vector is obtained by cloning a nucleotide sequence shown by SEQID No.3 in a sequence table to a prokaryotic expression vector.

6. The method of claim 4, wherein: the amino acid sequence of the recombinant protein antigen is SEQ ID No.2 in the sequence table.

7. The method of claim 4, wherein: the Escherichia coli competent cells were Rosetta (DE3) competent cells.

8. The production method according to any one of claims 4 to 7, characterized in that: the specific steps of immunizing animals by the recombinant protein antigen comprise: the recombinant protein antigen is emulsified and then used for immunizing animals, secondary immunization is carried out after 3 weeks of primary immunization, tertiary immunization is carried out after 2 weeks of interval, quaternary immunization is carried out after 2 weeks of interval of tertiary immunization, quinary immunization is carried out after 2 weeks of interval of quaternary immunization, and the antiserum titer is improved by 6 times of immunization at most.

9. A polyclonal antibody against EF-Tu protein, which is a non-lactococcus lactis surface adhesion protein, prepared by the preparation method according to any one of claims 4 to 8.

Technical Field

The invention belongs to the technical field of biotechnology, and particularly relates to a polyclonal antibody of a non-lactococcus lactis surface adhesion protein EF-Tu protein and a preparation method thereof.

Background

The non-lactose-solubilizing streptococcus LZMYFGM9 strain (the strain obtained in Chinese patent No.201210141827.5, the preservation number of the strain is CGMCC No.4227) is called as 'non-lactose-solubilizing streptococcus FGM strain' in the text, is 1 strain of lactic acid bacteria of fermentable astragalus which is separated from chicken intestinal tracts and cultured and domesticated by astragalus-containing culture, and has the characteristics of safety, high polysaccharide conversion rate, capability of inhibiting the damage of escherichia coli to organisms, promotion of intestinal mucosal immunity and the like. Research shows that the mediation of the lactobacillus surface adhesion protein and the exogenous substances thereof has important significance for improving the intestinal micro-ecological environment of animals, improving the immunity of organisms, enhancing the disease resistance and describing the biological action mechanism of the effective components of the traditional Chinese medicine. At present, the in vitro expression of the adhesion protein EF-Tu on the surface of the lactobacillus and the preparation of a specific antibody are not seen.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a polyclonal antibody of a non-lactococcus lactis surface adhesion protein EF-Tu protein and a preparation method thereof.

The invention provides an EF-Tu gene sequence of a non-lactococcus lactis surface adhesion protein, which is SEQ ID No.1 in a sequence table.

The invention provides an amino acid sequence of a non-lactococcus lactis surface adhesion protein EF-Tu protein, which is SEQ ID No.2 in a sequence table.

The invention provides an EF-Tu prokaryotic expression protein of a non-lactococcus lactis surface adhesion protein, which constructs a recombinant expression vector containing a nucleotide sequence shown by SEQ ID No.3 in a sequence table, and converts an escherichia coli competent cell into the prokaryotic expression protein.

The invention provides a preparation method of a polyclonal antibody of a non-lactococcus lactis surface adhesion protein EF-Tu protein, which comprises the following steps:

(1) constructing a recombinant expression vector containing a nucleotide sequence shown in SEQ ID No.3 in a sequence table, and transforming escherichia coli competent cells into the recombinant expression vector to obtain a recombinant protein antigen;

(2) immunizing animals with the recombinant protein antigen, and separating and purifying the serum to obtain the EF-Tu protein polyclonal antibody of the non-lactococcus lactis surface adhesion protein.

Preferably, the recombinant expression vector is obtained by cloning the nucleotide sequence shown by SEQ ID No.3 in the sequence table to a prokaryotic expression vector.

Preferably, the prokaryotic expression vector is PET-B2M.

In the present invention, prokaryotic expression vectors suitable for recombinant protein antigens, which are conventionally used in the art, may be used, and preferably, when the prokaryotic expression vector is PET-B2M, the expression effect of recombinant protein antigens is better.

Preferably, the amino acid sequence of the recombinant protein antigen is SEQ ID No.2 in the sequence table.

Preferably, the E.coli competent cell is a Rosetta (DE3) competent cell.

The type of the E.coli competent cells used in the present invention is not particularly limited as long as it is suitable for efficient expression of the recombinant expression vector in the cells, and preferably, the E.coli competent cells are Rosetta (DE3) competent cells in order to obtain a better expression effect.

Preferably, the specific steps of immunizing an animal with the recombinant protein antigen include: the recombinant protein antigen is emulsified and then used for immunizing animals, secondary immunization is carried out after 3 weeks of primary immunization, tertiary immunization is carried out after 2 weeks of interval, quaternary immunization is carried out after 2 weeks of interval of tertiary immunization, quinary immunization is carried out after 2 weeks of interval of quaternary immunization, and the antiserum titer is improved by 6 times of immunization at most.

Animals immunized include, but are not limited to, new zealand white rabbits, mice, and rats.

The invention also provides the non-lactococcus lactis surface adhesion protein EF-Tu protein polyclonal antibody prepared by the preparation method. The polyclonal antibody can specifically recognize a specific amino acid fragment SEQ ID No.2 in the EF-Tu protein, and is a specific antibody of the EF-Tu protein.

The invention determines the existence of EF-Tu participating in bacterial adhesion and permanent planting in FGM surface protein by LC-MS-MS technology, determines the gene sequence of the protein by using the FGM complete gene sequence, and further clones by using a PCR method to obtain a specific DNA fragment in the FGM bacterial EF-Tu protein to construct a recombinant expression vector. The EF-Tu recombinant protein expressed by pronucleus is used for immunizing a New Zealand white rabbit to obtain a specific polyclonal antibody. The polyclonal antibody prepared by the method provided by the invention can specifically recognize the EF-Tu protein of the FGM bacteria, but cannot recognize other proteins of the FGM bacteria, and has wide application in detection, functional identification and research of the EF-Tu protein.

Therefore, the FGM strain surface adhesion protein EF-Tu gene sequence, the amino acid sequence, the recombinant protein and the polyclonal antibody thereof have innovativeness, promote the application of probiotics and the protein thereof in animal feed additives, and have important significance for the action of reducing antibiotics in China.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:

FIG. 1 shows the result of the electron microscope analysis of FGM strain treated with alkaline LiCl.

FIG. 2 shows the results of SDS-PAGE electrophoresis of FGM surface protein and whole protein.

FIG. 3 shows the results of mass spectrometric identification.

FIG. 4 is a hydrophobicity analysis.

Fig. 5 is a disordered sequence analysis.

FIG. 6 is an antigenic analysis.

FIG. 7 shows homology analysis.

FIG. 8 is a domain analysis.

FIG. 9 shows the amplification of the EF-Tu gene of FGM strain.

FIG. 10 is a PCR electrophoretic analysis chart of positive transformants.

FIG. 11 is an SDS-PAGE analysis of EF-Tu protein purification.

Figure 12 is a graph of antiserum titers.

FIG. 13 is a diagram of the rabbit anti-EF-Tu protein polyclonal antibody purification analysis.

FIG. 14 shows the result of Western blot identification of recombinant proteins.

FIG. 15 shows the identification result of the FGM surface protein EF-Tu.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.