CN112979769B

CN112979769B - Amino acid sequence, protein, preparation method and application thereof

Info

Publication number: CN112979769B
Application number: CN202110252533.9A
Authority: CN
Inventors: 聂金梅; 何丽娟; 刘秀贵; 胡志强; 李燚; 汤茂和; 徐亮
Original assignee: Zhuhai Lihe Medical Diagnosis Products Co ltd
Current assignee: Zhuhai Lihe Medical Diagnosis Products Co ltd
Priority date: 2021-03-08
Filing date: 2021-03-08
Publication date: 2023-01-31
Anticipated expiration: 2041-03-08
Also published as: CN112979769A

Abstract

The invention relates to the technical field of biology, in particular to an amino acid sequence, a protein, a preparation method and an application thereof. The invention provides the application of the amino acid sequence in preparing mycoplasma pneumoniae antigens or products containing the mycoplasma pneumoniae antigens, wherein the amino acid sequence comprises (a) an amino acid sequence shown as SEQ ID No.1 or (b) the amino acid sequence has homology of more than 88 percent with the amino acid sequence shown as SEQ ID No. 1; and the amino acid sequence does not comprise the sequence shown in SEQ ID No. 2. The protein encoded by the amino acid sequence shown in SEQ ID No.1 or the equivalent functional sequence thereof provided by the invention has no transmembrane region, is easy to produce, and has high sensitivity and strong specificity.

Description

Amino acid sequence, protein, preparation method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an amino acid sequence, a protein, a preparation method and an application thereof.

Background

Mycoplasma Pneumoniae (MP) is one of the important pathogens of human respiratory tract infection, and infection with Mycoplasma pneumoniae can cause mild pharyngolaryngitis, bronchitis, severe interstitial pneumonia (primary atypical pneumonia), and complications of nervous system and cardiovascular system.

The current routine Mycoplasma pneumoniae detection methods include culture methods, PCR detection, and serological diagnostics. The growth of the mycoplasma pneumoniae is slow, and the culture method cannot meet the requirement of clinical rapid diagnosis. Although the PCR method improves the sensitivity of detection, it requires a standard operation and strict quality control, otherwise it is liable to non-specific reaction. The common clinical methods are serological diagnosis, the antigens used by the serological diagnostic kits commonly used at home and abroad are mycoplasma pneumoniae holobacteria or cell membrane fragment components thereof, and the specificity is low because sugar ester components in the mycoplasma pneumoniae holobacteria or the cell membrane fragment components thereof have cross reaction with partial bacteria and human tissues.

At present, the commonly used standardized kits in China are a Japanese imported SERODIA-MYCO II kit and a U.S. imported EIA detection kit, the antigen components are mainly mycoplasma pneumoniae cell membrane fragment antigens, and the kits have high cost and are easy to generate non-specific cross reaction.

A key problem in preparing a mycoplasma pneumoniae immunodiagnosis kit is that a detection antigen with high purity and high immunogenicity must be obtained, and a mycoplasma pneumoniae antigen with high specificity is urgently searched for being used for serological mycoplasma pneumoniae diagnosis.

The P30 protein is an important adhesion protein anchored on the MP cell membrane, comprises 275 amino acid residues, has the relative molecular mass of about 29.7Kd, and has the gene sequence with the full length of 825bp. Studies have shown that the P30 protein can stimulate host immune response, and is another important adhesin and immunogen in addition to the adhesion protein P1. The P30 antigen is cloned and expressed by Varshney et al (Varshney AK, chaudhry R, kabra S K, et al cloning, expression, and immuNological characterization of the P30 protein of Mycoplasma pnuemonia [ J ]. Clin Vaccine Immunol.2008,15 (2): 215-220.), but the P30 antigen adopts a wild-type gene, and the expression level is low, thereby limiting the application of the P30 antigen in diagnosis and Vaccine development. The adhesion protein P30 is adhered to a cell membrane, and a part of polypeptide sequence is embedded into a hydrophobic region of the membrane, so that the lipid solubility is strong; the recombinant Escherichia coli is induced and expressed in an inclusion body, the purification process of the membrane protein is complicated, the yield is low, the industrial production is difficult to realize, and the sensitivity and the specificity of the adhesion protein P30 are to be improved.

The present invention has been made based on the above analysis.

Disclosure of Invention

The invention mainly aims to provide an amino acid sequence with a specific recognition function on a mycoplasma pneumoniae antibody, and a protein which is easy to express and purify, has high sensitivity and specificity and is suitable for industrial production is constructed by taking the amino acid sequence as a core so as to solve the technical problem in the detection of the mycoplasma pneumoniae antibody at present.

The second purpose of the invention is to protect the polypeptide coded by the amino acid sequence so that the polypeptide can meet the requirement of industrial production.

The third purpose of the invention is to protect the protein coded by the amino acid sequence so that the protein can meet the requirement of industrial production.

It is a fourth object of the present invention to provide a nucleic acid molecule encoding the above protein and a biomaterial constructed by further using the nucleic acid molecule, thereby achieving high expression of the above protein.

The fifth object of the present invention is to provide a method for producing the above protein, so as to obtain the above protein.

The sixth purpose of the present invention is to provide the use of the above protein, nucleic acid molecule or biomaterial in the preparation of a pneumonia vaccine product or a pneumonia mycoplasma detection product.

The last object of the present invention is to provide a reagent for detecting mycoplasma pneumoniae which enables detection of mycoplasma pneumoniae for non-diagnostic purposes or non-therapeutic purposes using a corresponding detection method.

In order to solve the technical problems and achieve the purpose, the invention provides the following technical scheme:

in a first aspect, the present invention provides an amino acid sequence comprising any one of:

(a) An amino acid sequence shown as SEQ ID No. 1;

(b) The amino acid sequence has more than 88 percent of homology with the amino acid sequence shown in SEQ ID No. 1;

and, the amino acid sequence does not comprise the sequence shown in SEQ ID No. 2.

In an alternative embodiment, the amino acid sequence is as set forth in SEQ ID No. 3.

In a second aspect, the present invention provides a polypeptide comprising an amino acid sequence as described in the preceding embodiments.

In a third aspect, the present invention provides a protein comprising an amino acid sequence according to the previous embodiment.

In an alternative embodiment, the protein comprises a first protein fragment encoded by an amino acid sequence as described in the previous embodiments, and at least the N-terminus of the first protein fragment has a protective modification.

Preferably, the protective modification comprises attachment of a protective protein.

Preferably, the first protein fragment has a protective protein attached to both the N-terminus and the C-terminus.

Preferably, a second protein fragment is connected between the N end of the first protein fragment and the protective protein, and the amino acid sequence of the second protein fragment is shown as SEQ ID No. 4.

In alternative embodiments, the protective protein comprises at least one protein tag, or a protein with the same protective function after the protein tag is modified by an amino acid sequence; the amino acid sequence modification comprises the protein which is obtained by substituting and/or deleting and/or adding at least one amino acid residue in the amino acid sequence of the protein tag and has homology of more than 88 percent with the amino acid sequence of the protein tag.

Preferably, the number of the protein tags or the proteins with the same function after the protein tags are modified by amino acid sequences is 1 to 3, and more preferably 3.

In alternative embodiments, the C-terminal linked protection protein of the first protein fragment comprises one or more of a6 xhis tag, an S-tag, a TrxA tag, a GST tag, an MBP tag, a SUMO tag, or a NusA tag; the protective protein connected with the N end of the first protein fragment comprises one or more than two of an S-tag label, a TrxA label, a GST label, an MBP label, a SUMO label or a NusA label; or the protective protein connected with the N end of the first protein fragment comprises a6 XHis tag and one or more of an S-tag, a TrxA tag, a GST tag, an MBP tag, an SUMO tag or a NusA tag.

In alternative embodiments, the first protein fragment and the protective polypeptide, the first protein fragment and the protective protein, the first protein fragment and the second protein fragment, or the protective protein are linked by a linker peptide.

Preferably, the connecting peptide comprises one of a short peptide LQ, a rigid linker or a multiple cloning site, and the multiple cloning site is shown as SEQ ID No. 6.

Preferably, the amino acid sequence of the rigid linker comprises (EAAAK) n, wherein n is 2 or 3.

Preferably, the protein comprises a TrxA tag, a6 xHis tag, an S-tag and a first protein fragment which are connected in sequence from the N end to the C end, a first connecting peptide is connected between the TrxA tag and the 6 xHis tag, a second connecting peptide is connected between the 6 xHis tag and the S-tag, a third connecting peptide is connected between the S-tag and the antigen protein, the amino acid sequence of the first connecting peptide is shown in SEQ ID No.5, the amino acid sequence of the second connecting peptide comprises a thrombomin enzyme cutting site and an enzyme cutting site protection amino acid residue, and the amino acid sequence of the third connecting peptide comprises a multiple cloning site as shown in SEQ ID No. 8.

Preferably, the protein consists of an MBP tag, a6 XHis tag and a first protein fragment which are sequentially connected from an N end to a C end, a fourth connecting peptide is connected between the MBP tag and the 6 XHis tag, a fifth connecting peptide is connected between the 6 XHis tag and the first protein fragment, the amino acid sequence of the fourth connecting peptide comprises a TEV enzyme cutting site and an enzyme cutting site protection amino acid residue, as shown in SEQ ID No.7, and the amino acid sequence of the fifth connecting peptide comprises a BamH1 enzyme cutting site, such as GS.

Preferably, the protein consists of an S-tag, a first protein fragment and a6 XHis tag which are sequentially connected from an N end to a C end, a sixth connecting peptide is connected between the S-tag and the first protein fragment, a seventh connecting peptide is connected between the first protein fragment and the 6 XHis tag, the amino acid sequence of the sixth connecting peptide comprises a polyclonal site, as shown in SEQ ID No.6, and the seventh connecting peptide comprises an Xho1 enzyme cutting site, such as LE.

Preferably, the protein consists of an S-tag and a first protein fragment which are sequentially connected from N end to C end, and are connected through a multiple cloning site.

In a fourth aspect, the present invention provides a nucleic acid molecule for editing a protein according to the previous embodiments, said nucleic acid molecule comprising a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No. 1.

Preferably, the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No.1 comprises the nucleic acid sequence shown in SEQ ID No. 9.

In a fifth aspect, the present invention also provides a biological material comprising a nucleic acid molecule according to the previous embodiment, the biological material comprising any one of (a) to (d):

(a) An expression cassette;

(b) A recombinant vector;

(c) Recombinant eukaryotic cells;

(d) Recombinant prokaryotic cells.

Preferably, the recombinant vector uses plasmid pET32a as a vector.

Preferably, the recombinant prokaryotic cell selects escherichia coli as a host cell, and further preferably E.

In a sixth aspect, the present invention also provides a method for producing a protein according to the foregoing embodiment, wherein the method comprises expressing and translating a nucleic acid molecule according to the foregoing embodiment or a biological material according to the foregoing embodiment to obtain a protein product, purifying and concentrating the protein product to obtain the protein.

In a seventh aspect, the present invention also provides an application of the protein, the nucleic acid molecule, the biological material or the protein obtained by the preparation method of the foregoing embodiments in the preparation of a pneumonia vaccine product or a pneumonia mycoplasma detection product.

In an eighth aspect, the present invention also provides a reagent for detecting mycoplasma pneumoniae, wherein the reagent for detecting mycoplasma pneumoniae comprises the protein described in the foregoing embodiment, the protein encoded by the nucleic acid molecule described in the foregoing embodiment, the protein expressed by the biological material described in the foregoing embodiment, or the protein prepared by the preparation method described in the foregoing embodiment.

In a ninth aspect, the present invention also provides a method for detecting mycoplasma pneumoniae by using the reagent for detecting mycoplasma pneumoniae described in the previous embodiments, wherein the method for detecting mycoplasma pneumoniae comprises chemiluminescence, ELISA, a rapid colloidal gold detection method, an agar diffusion method, an agglutination method or an immunoblotting method.

The protein encoded by the amino acid sequence shown in SEQ ID No.1 or the equivalent functional sequence thereof does not have a transmembrane region and has the characteristics of high sensitivity and strong specificity.

The invention provides an amino acid sequence shown as SEQ ID No.1 or a sequence with equivalent specificity recognition function to a mycoplasma pneumoniae antibody, and a polypeptide or protein coded by the amino acid sequence, which is easier to produce compared with P30 and has higher sensitivity and specificity compared with the whole mycoplasma pneumoniae bacterium.

The protein is subjected to protective modification, at least the N end of the protein is connected with the protective polypeptide or the protective protein, and the modified protein shows good solubility in cells, is not easy to degrade and does not need protein renaturation, so that the protein is closer to the natural structure of the protein, has better performance in downstream experimental application, and is more suitable for popularization and citation.

The invention further provides a mycoplasma pneumoniae detection reagent and a mycoplasma pneumoniae detection kit containing the amino acid sequence or the protein, and the market demand is met.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the comparison of the sequence of SEQ ID NO.11 with the sequence of SEQ ID NO.9 in an embodiment of the present invention;

FIG. 2 shows the comparison of the sequence of SEQ ID NO.1 with the sequence of SEQ ID NO.10 in an embodiment of the present invention;

FIG. 3 shows the comparison of the sequence of SEQ ID NO.13 with the sequence of SEQ ID NO.9 in an embodiment of the present invention;

FIG. 4 shows the comparison of the sequence of SEQ ID NO.12 with the sequence of SEQ ID NO.1 in an embodiment of the present invention;

FIG. 5 shows the predicted result of P30 transmembrane region;

FIG. 6 shows the predicted result of the P30B1 transmembrane region;

FIG. 7 is a P30B1 antigenicity prediction result;

FIG. 8 shows the CAI values after codon optimization of the P30B1 nucleic acid sequence in example 1;

in FIG. 9, a is an SDS-PAGE pattern of TrxA-6 XHis-Stag-P30B 1 purified by the Ni ion affinity chromatography column in example 1; b is an SDS-PAGE picture of purified TrxA-6 XHis-Stag-P30B 1 by Q-HP ion exchange chromatography in example 1; c is SDS-PAGE picture of the sample after the TrxA-6 xHis-Stag-P30B 1 protein purification and concentration in the example 1; d is an SDS-PAGE picture of the TrxA-6 XHis-Stag-P30B 1 protein after being subjected to enzyme digestion by Thrombin in example 1;

in FIG. 10, a is an SDS-PAGE pattern after purification of the S-tag-P30B1 protein in example 1; b is the SDS-PAGE picture after purification of the S-tag-P30B1-6 XHis protein in example 2; c is SDS-PAGE picture of MBP-6 x his-P30B1 protein purified by the Ni ion affinity chromatographic column in example 4; d is an SDS-PAGE pattern of a sample obtained by desalting and concentrating the MBP-6 XHis-P30B 1 protein in example 4; e is an electrophoresis result chart of the sample subjected to TEV enzyme digestion in example 4; f is the SDS-PAGE pattern of the purified P1 fragment-LQ-P30B 1 fusion protein obtained in example 5.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, as presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

It should be noted that: like reference numbers and letters refer to like items in the following figures, and thus, once an item is defined in one figure, it need not be further defined and explained in subsequent figures.

The terms "first," "second," "third," and the like as used herein are used solely to distinguish one from another and are not intended to indicate or imply relative importance.

It should also be noted that, unless expressly specified or limited otherwise, the term "coupled" is to be construed broadly, as meaning, for example, directly coupled or indirectly coupled through intervening media. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

The homology for the sequences described in the present invention is explained below: the person skilled in the art can obtain the protein similarity information by comparing the protein sequence similarities, analyze the sequence conservation types of homologous proteins in the evolution process and predict the possible protein structural domains. Sequence homology detection can identify homologous proteins or genes by detecting too high a degree of similarity: when the similarity of two sequences exceeds that expected by chance, we conclude that there is homology between the two sequences. The homology calculation formula of the present invention is the percentage of the number of identical amino acids to the total number of amino acids of the full-length sequence or the percentage of the number of identical bases to the total number of bases of the full-length sequence.

Thus, for the amino acid sequence of SEQ ID NO.1 according to the invention having a homology of 88% or more, the following are exemplified:

for example one

The homology analysis of the protein sequence SEQ ID NO.1 coded by the nucleic acid sequence SEQ ID NO.9 and the protein sequence SEQ ID NO.10 coded by the SEQ ID NO. 11.

The same base number of the SEQ ID NO.11 sequence and the SEQ ID NO.9 is 521, the total length of the base sequence of the SEQ ID NO.11 is 579, the similarity is 88.67% (525/579), the nucleic acid sequence has high consistency, the SEQ ID NO.11 sequence and the two sequences of the SEQ ID NO.9 can be deduced to have homology, the comparison result of the nucleic acid sequences is shown in FIG. 1, wherein in the two sequences for comparison, the upper row is the sequence of the SEQ ID NO.9, and the lower row is the sequence of the SEQ ID NO. 11.

The number of identical amino acids of SEQ ID NO.1 and SEQ ID NO.10 is 177, the total length of the amino acid sequence of SEQ ID NO.10 is 192, the similarity is 92.19% (177/192), the amino acid sequences have high consistency, and the deduced homology of the two sequences of SEQ ID NO.1 and SEQ ID NO.10 can be deduced, the amino acid sequence alignment result is shown in FIG. 2, wherein in the two rows of sequences to be aligned, the upper row is the sequence of SEQ ID NO.1, and the lower row is the sequence of SEQ ID NO. 10.

Example II

The homology analysis of the protein sequence SEQ ID NO.1 coded by the nucleic acid sequence SEQ ID NO.9 and the protein sequence SEQ ID NO.12 coded by the nucleic acid sequence SEQ ID NO. 13.

The number of bases in the sequence of SEQ ID NO.13 identical to that of the sequence of SEQ ID NO.9 is 267, the full length of the sequence of SEQ ID NO.13 is 600 bases, and the similarity of the sequence of SEQ ID NO.13 to the sequence of SEQ ID NO.9 is 44.27%

(267/603), the nucleic acid sequence does not have identity, and it can be concluded that the sequence of SEQ ID NO.13 has no homology with the two sequences of the sequence of SEQ ID NO. 9. The results of the nucleic acid sequence alignment are shown in FIG. 3, wherein the upper row of the two rows of sequences to be aligned is the sequence of SEQ ID NO.9, and the lower row is the sequence of SEQ ID NO. 13.

The same amino acid number of the sequence SEQ ID NO.1 and the sequence SEQ ID NO.12 is 24, the full-length amino acid number of the sequence SEQ ID NO.12 is 200, the similarity of the sequence SEQ ID NO.1 and the sequence SEQ ID NO.12 is 12% (24/200), the sequence SEQ ID NO.1 and the sequence SEQ ID NO.12 have no consistency, the sequence SEQ ID NO.1 and the two sequences of the sequence SEQ ID NO.12 do not have homology, the amino acid sequence alignment result is shown in FIG. 4, wherein in the two rows of sequences for comparison, the upper row is the sequence SEQ ID NO.1, and the lower row is the sequence SEQ ID NO. 12.

In the present invention, since differences in structure due to chemical modifications of bases, nucleotides, ribonucleotides, deoxyribonucleotides, amino acids, and amino acid residues, for example, fluorine modification of nucleotides, demethylation modification of nucleotides, replacement of DNA or RNA with PNA, and the like, are considered to be the same, it is considered that chemical modifications of bases, nucleotides, ribonucleotides, deoxyribonucleotides, amino acids, and amino acid residues do not affect homology.

In the invention, the protein which is generated by performing a fusion experiment on the fragment P30B1 and has a specific recognition function of more than 88% on the mycoplasma pneumoniae antibody still falls into the protection scope of the invention.

The mycoplasma pneumoniae antibody in the invention refers to a protein, polypeptide or other substances which can generate specific binding with mycoplasma pneumoniae antigen or polypeptide, and can also be called as an anti-mycoplasma pneumoniae antibody.

The mycoplasma pneumoniae antigen in the invention refers to protein or polypeptide which can generate an antigen-antibody specific structure with a mycoplasma pneumoniae antibody.

The drawbacks that exist for the specific detection of the P30 protein directly in mycoplasma pneumoniae are illustrated below:

a fat-soluble transmembrane region fragment exists in the P30 protein, and through bioinformatics software analysis, as shown in figure 5, 70 amino acids (shown as B in figure 5) at the N end of the P30 protein are transmembrane regions (transmembrane), the partial polypeptide sequence is embedded into a hydrophobic region of the membrane, the fat solubility is strong, the partial polypeptide sequence exists in an inclusion body after being induced and expressed by escherichia coli recombinant bacteria, the purification process of the membrane protein is complicated, and experimental results show that the sensitivity and the specificity of the P30 protein cannot meet application requirements.

The invention firstly selects 5 fragments from P30 antigen: the sensitivity and specificity of the fragment P30B1 (amino acid residues: 98-274), the P30 fragment 1 (amino acid residues: 118-274), the P30 fragment 2 (amino acid residues: 107-161), the P30 fragment 3 (amino acid residues: 98-204) and the P30 fragment 4 (amino acid residues: 28-204) are obviously higher than those of other four fragments and the P30 protein as shown in Table 14 (shown in the specification) by constructing the fragments on a pET32a vector, fusing a label TrxA +6 xHis + S-tag at the N terminal and detecting the fragments by an ELisa method after expression and purification. The P30 fragment 1 is a shortened sequence of P30B1, and has 88.4% homology with the P30B1 protein, so that it is inferred that the P30 fragment 1 has the effect of enhancing sensitivity and specificity compared with the P30 fragment having 88% or more homology with the P30B1 protein.

The P30B1 protein protected by the invention is a periplasmic space expression protein which is further derived after a P30 protein transmembrane region is removed, the protein does not have the transmembrane region, as shown in figure 6, the protein directly exists in a periplasmic space after being expressed, the oxidation environment of the periplasm is favorable for the correct folding of the protein, and in the process of transferring to the periplasm, a signal peptide is sheared in cells and is more likely to generate the natural N tail end of the target protein, so that the protein is closer to the natural structure of the protein and has better performance in downstream experimental application. The results of antigenicity prediction of the P30B1 protein show that the Score mean value of the whole sequence is more than 5, and the antigenicity is better (the results show that the Score value is more than 3, and the P30B1 protein can be developed into independent antigenic fragments), as shown in FIG. 7.

In addition, in order to enhance the stability of the structure of the protein P30B1 encoded by the truncated gene, at least the N terminal of the protein P30B1 is subjected to protective modification, the optional protective modification comprises connection of protective protein, and the optional protective protein comprises more than one of an S-tag, a TrxA tag, a GST tag, an MBP tag, a SUMO tag or a NusA tag, or more than one of a6 xHis tag, an S-tag, a TrxA tag, a GST tag, an MBP tag, an SUMO tag or a NusA tag.

However, it should be noted that when the N-terminal of the P30B1 sequence is linked to only 6 × His, the protein P30B1 is rapidly degraded and extremely unstable, indicating that the N-terminal linked to only 6 × His does not contribute to maintaining the structural integrity of the N-terminal of the protein.

In the embodiment of the invention, trxA-6 xhis-S-tag-P30B 1 protein, MBP-6 xhis-P30B 1 protein, S-tag-P30B1-6 xhis protein, fusion protein P1 fragment-linker-P30B 1, P1 fragment protein, and P30 protein are prepared, and the application of the proteins and mycoplasma pneumoniae holosomatic antigen (commercially available) is tested, and the results show (see the following examples): the sensitivity and specificity of the P30B1 fusion protein containing the protein tag are superior to those of a whole bacterial antigen and a P30 protein, the probability of false positive and false negative of the protein P30B to the detection of mycoplasma pneumoniae is greatly lower, the sensitivity and specificity of a fusion protein P1 fragment-linker-P30B 1 are respectively 94.00 percent and 98.04 percent, and the sensitivity and specificity are superior to those of the P30B1 fusion protein containing the protein tag and the P1 fragment protein.

Some embodiments of the invention are described in detail below with reference to the accompanying drawings. The embodiments and features of the embodiments described below can be combined with each other without conflict.

Example 1:

the invention aims to optimize synthesis of genes and construct recombinant vectors

Analyzing the whole amino acid sequence of the adhesion protein P30 of the mycoplasma pneumoniae (FH strain) by utilizing ProtScale software, wherein the amino acid sequence is 275 in total, the protein is adhered to a cell membrane, and part of polypeptide sequence is embedded into a hydrophobic region of the membrane, so that the fat solubility is strong; constructing an escherichia coli recombinant strain, finding that the escherichia coli recombinant strain exists in an inclusion body after induction expression, and having complex purification process of membrane protein, low yield and difficulty in realizing industrial production; in order to realize the soluble expression of the antigenic fragment, firstly, the transmembrane segment is knocked out at the gene level, the amino acid sequence of the transmembrane segment is shown as SEQ ID No.14, the amino acid fragment P30B1 (amino acid residues: 98-274) with strong solubility and antigenicity is reserved, and in order to enhance the stability of the protein structure coded by the truncated gene, amino acid labels are fused at the C terminal and/or the N terminal of the truncated gene, so that the aim of improving the stability and the solubility of the protein is fulfilled, and the purification and the preparation of the high-purity protein are facilitated.

The method comprises the following specific steps:

1.1 construction of recombinant vector P30B1-pET32a

The gene sequence of the 98-274aa fragment encoding the P30B1 protein is subjected to codon optimization aiming at the codon preference of the large intestine so as to improve the expression level of the target protein in the large intestine, the optimization process comprises optimizing rare codons which reduce the translation efficiency and even prevent the translation in the original gene sequence into codons preferred by the large intestine, the optimized CAI is improved to 0.81, as shown in figure 8, simultaneously, the GC content of the optimized gene sequence prolongs the half life of mRNA, breaks a stem-loop structure which influences ribosome binding and mRNA stability, and in addition, the following cis-acting elements are screened and optimized: coli _ RBS (AGGAGG), polyT (TTTTTT), polyA (AAAAAAA), chi _ sites (GCTGGTGG), T7 Cis (ATCTGTT) and SD _ like (GGRGGT), and obtaining an optimized gene fragment SEQNO.9 by adopting a total synthesis gene method, constructing the optimized gene fragment SEQNO.9 between a multiple cloning site BamH1 and an Xho1 of a Pet32a vector, and entrusting Nanjing Kingsry Biotech limited company to synthesize to obtain a recombinant vector P30B1-pET32a.

1.2 construction of recombinant plasmid MBP-P30B1-pET28a

Carrying out double enzyme digestion on the P30B1-pET32a plasmid obtained in the step 1 and a vector MBP-pET28a (152 ng/. Mu.l) stored in the experiment by using BamH1 and Xho1, carrying out enzyme digestion for 0.5h at 37 ℃, directly using the vector MBP-pET28a subjected to double enzyme digestion for subsequent experiments, carrying out agarose gel electrophoresis on the enzyme digestion product of the P30B1-pET32a plasmid obtained in the step 1, carrying out gel digestion, purifying and recovering a target gene fragment with the size of about 500bp, and measuring the nucleic acid concentration by using a nanodrop instrument to be 65 ng/. Mu.l respectively.

And (2) taking 7 mu l of the enzyme digestion product of the P30B1-pET32a plasmid obtained in the step (1), 3 mu l of the enzyme digestion product of the MBP-pET28a, and 10 mu l of 2 x Infusion-clone mix (Takara), connecting through multiple cloning sites at 37 ℃, cloning for 0.5h, transforming the seamless cloning product into Top10 competent cells, culturing the recombinant bacteria with correct sequencing, and extracting the MBP-P30B1-pET28a plasmid.

1.3 induced expression and purification of TrxA-6 × his-S-tag-P30B1 and S-tag-P30B1 proteins

Adding purified water into the MBP-P30B1-pET32a plasmid dry powder obtained in the step 1 to dissolve until the solubility is 40 ng/mu l, taking 2 mu l to add into 100 mu l of escherichia coli E.coli Rosetta (DE 3) competent cells, respectively coating LB culture plates containing 100 mu g/ml of ampicillin and 50 mu g/ml of chloramphenicol, and culturing for 14-16h at 37 ℃ overnight; and (3) selecting a single colony of the positive recombinant bacteria, inoculating the single colony into 5ml of LB liquid culture medium containing the same antibiotics, and culturing overnight for 14-16 h.

Inoculating the overnight cultured bacterial liquid into 15ml LB culture medium containing the same antibiotic according to the inoculation amount of 1%, culturing at 37 ℃ and 250rpm until the OD600 of the bacterial liquid is 1.0-1.3, adding IPTG with the final concentration of 1.0mM, and inducing at 25 ℃ and 250rpm for 3-4 h.

The cells were collected by centrifugation at 1.4, and 8mL of lysine buffer (20 mM PBS +500mM NaCl, pH 7.4) was added to ice water to resuspend the solid until no clump of cells were visible to the naked eye.

1.5 ultrasonic disruption: and (4) breaking the bacteria according to the conditions of ultrasonic power of 400W, ultrasonic time of 4s and stopping for 3s, and circulating for 50 times until the bacteria liquid is clear and transparent. Centrifuging at 4 deg.C and 12000rpm for 30min after the ultrasound treatment, separating supernatant and precipitate, and filtering the supernatant with 0.22 μm membrane; the supernatant and the precipitate were each left to be 20. Mu.l, subjected to SDS-PAGE, and subjected to a small-scale expression test to screen recombinant bacteria that can express the target protein in a soluble form.

1.6 pretreatment of Ni ion affinity chromatography column: 5 column volumes were washed with buffer B (20mM PBS,1M imidazole, pH 7.4) and then 10 column volumes were equilibrated with buffer A.

1.7 loading: the filtered supernatant was subjected to Ni column affinity chromatography at a flow rate of 2ml/min, and the flow-through was collected in a clean vial, designated FT, and 20. Mu.l was left.

1.8, cleaning: after the flow-through was complete, the column was flushed with lysis buffer (20mM PBS +500mM NaCl, pH 7.4) until the baseline leveled off (approximately 10 column volumes) and the flow rate was unchanged.

1.9 elution: the imidazole concentration was used sequentially at 20mM,40mM,80mM,160mM,250mM, and 1M, respectively, and the eluate was collected and 20. Mu.l of the sample was retained.

1.10 the samples retained in the above steps are added with 5. Mu.l of 5xloading buffer to all the EP tubes in the order of elution, and SDS-PAGE is carried out in the order, and the result is shown in FIG. 9 a.

1.11 pretreatment of Q-HP ion exchange chromatography column: the 10 column volumes were washed with the high salt buffer and then 10 column volumes were equilibrated with the low salt buffer.

1.12 loading: the sample purified by Ni affinity chromatography was diluted to 10ml with Buffer A (20mM PBS, pH 7.4), purified by Q-HP ion exchange chromatography at a flow rate of 1ml/min, and the flow-through was collected in a clean vial.

1.13 elution: elution was performed with a gradient of Buffer C (20mM PBS +1M NaCl, pH 7.4) at 4%, 6%, 8%, 10%, 15%, 20%, 50% and 100%, and the eluates were collected in peak tubes and 20. Mu.l were retained.

1.14 electrophoresis: SDS-PAGE (4% -20% gradient gel) was performed in the order of elution, and the results are shown in FIG. 9 b. TrxA-6 × his-S tag-P30B1 protein was purified and concentrated as shown in FIG. 9 c.

1.15 60mg of TrxA-6 × his-S-tag-P30B1 protein was added to 120U Thrombin and digested at 20 ℃ overnight.

1.16 the digested sample was purified by Ni ion affinity chromatography column as above 6-9, and the electrophoresis result after purification is shown in FIG. 9 d.

1.17 according to the result of the electrophoresis chart, collecting the flow-through liquid (FT), concentrating and desalting by using an ultrafiltration tube, purifying by using a Q-HP ion exchange chromatography column, and operating the operations from 10 to 13 to obtain the purified S-tag-P30B1 protein, wherein as shown in FIG. 10a, the S-tag-P30B1 protein can still keep the P30B1 protein sequence complete, and the N end of the P30B1 is not degraded, so that the S-tag-P30B1 protein has good stability.

Example 2

In this example, protein S-tag-P30B1-6 XHis was prepared by the following steps:

2.1 construction of fusion protein S-tag-P30B1-6 XHis recombinant vector

Using S-tag-P30B1-6 XHis-pET 32a recombinant plasmid as a template, connecting the S-tag and the P30B1 through a multiple cloning site, connecting the P30B1 and the 6 XHis through an Xho1 enzyme cutting site, then designing a primer, obtaining an S-tag-P30B1-6 XHis gene fragment by adopting a PCR method, cutting glue, purifying and recycling, determining the nucleic acid concentration to be 73 ng/mu l, constructing the nucleic acid concentration to a linearized vector pET28a by adopting a seamless cloning technology, obtaining 3 mu l of an S-tag-P30B1-6 XHis gene product, obtaining 2 mu l of a linearized pET28a vector, obtaining 2 mu l of 2 × ultra clone expressmix 5 mu l (purchased from Nanjing Nuozu Bio-Tech Co., ltd.), 37 ℃ and 30min; adding 10 mu l of seamless cloning ligation product into 100 mu l of homemade Top10 competent cells, carrying out ice bath for 30min, carrying out water bath for 88s at 42 ℃, then putting the cells into ice water for 2-5min, adding 700 mu l of nonreactive LB liquid culture medium, carrying out shaking culture at 37 ℃ and 250rpm for 1h, centrifuging (4500 rpm and 5 min), discarding 600 mu l of supernatant, carrying out heavy suspension on the cells, taking 100 mu l of bacterial liquid, coating the bacterial liquid on an LB solid culture medium containing 100 mu g/ml ampicillin, pouring the dried bacterial liquid into a constant temperature incubator after drying, carrying out overnight culture at 37 ℃ (14-16 h), picking 3 monoclonal transformants on the next day, respectively inoculating the transformants into 4ml of LB liquid culture medium containing 100 mu g/ml ampicillin, carrying out shaking culture at 37 ℃ and 250rpm for 6-8h, and sequencing to verify whether the recombinant vector construction is correct.

2.2 expression and purification of the protein S-tag-P30B1-6 XHis

Extracting the bacterial liquid with correct sequencing, measuring the concentration to be 164 ng/mul, adding 1 mul into 100 mul of self-made Rosetta (DE 3) competent cells, carrying out ice bath for 30min, carrying out water bath at 42 ℃ for 88s, putting into ice water for 2-5min, adding 700 mul of LB liquid culture medium without resistance, carrying out shaking culture at 37 ℃ and 250rpm for 1h, centrifuging (4500 rpm and 5 min), discarding 600 mul of supernatant, carrying out heavy suspension on the bacterial liquid, taking 100 mul of bacterial liquid, coating the bacterial liquid on LB solid culture medium containing 100 mul/ml ampicillin and 50 mul g/ml chloramphenicol, pouring the dried bacterial liquid into a constant temperature incubator, carrying out overnight culture at 37 ℃ (14-16 h), carrying out next day, selecting 3 monoclonals, respectively inoculating into 15ml LB liquid culture medium containing 100 mul g/ml ampicillin and 50 mul g/ml chloramphenicol, adding IPTG at 37 ℃ and 250rpm, carrying out shaking culture for 4h, adding IPTG at the final concentration to induce expression at 1mM, carrying out induction for 4h, discarding supernatant, centrifuging at 4500rpm, and collecting supernatant.

The steps of disrupting the bacteria with ultrasound and purifying the protein were the same as in example 1, and the electrophoresis result of S-tag-P30B1-6 XHis after purification is shown in FIG. 10B.

Example 3

This example differs from example 1 only in that the P30B1 sequence fragment in example 1 is substituted for P30 fragment 1 (amino acid residues: 118-274).

Example 4

This example provides a method for preparing protein MBP-6 XHis-P30B 1, comprising the following steps:

the construction of MBP-6 XHis-P30B 1-pET28a recombinant plasmid, wherein MBP was linked to 6 XHis via the enzyme-cutting site protection base FEGS and TEV enzyme-cutting site ENLYFQS in sequence, and 6 XHis and P30B1 were linked via the enzyme-cutting site BamH1, and then the recombinant plasmid was transformed and spread on LB culture plates containing 50. Mu.g/ml kanamycin and 50. Mu.g/ml chloramphenicol, and the other experimental procedures were the same as in example 1. Purifying with Ni ion affinity chromatography column and Q-HP ion exchange chromatography column to obtain MBP-6 XHis-P30B 1 protein, with the results shown in FIG. 10c and FIG. 10 d; in order to study the stability of the P30B protein, 6mg of MBP-6 XHis-P30B 1 protein is added with 600. Mu.g of TEV enzyme, the enzyme digestion is carried out at 4 ℃ overnight, 20. Mu.l of the sample after the enzyme digestion is added with 5. Mu.l of loading buffer, and SDS-PAGE is carried out, and the result is shown in figure 10e, and the result in figure 10e shows that only protein bands of MBP and TEV enzyme exist in the sample after the TEV enzyme digestion, and no protein band of 6 XHis-P30B 1 exists, which indicates that the P30B1 protein is easy to degrade and can not stabilize the protein structure with biological activity when only one 6 XHis tag is fused at the N terminal.

Comparative example 1

This comparative example differs from example 1 only in that the P30B1 sequence fragment in example 1 is replaced by P30 fragment 2 (amino acid residues: 107-161).

Comparative example 2

This comparative example differs from example 1 only in that the P30B1 sequence fragment in example 1 is replaced by P30 fragment 3 (amino acid residues: 98-204).

Comparative example 3

This comparative example differs from example 1 only in that the P30B1 sequence fragment in example 1 is replaced by P30 fragment 4 (amino acid residues: 28-204).

Experimental example 1

The sensitivity and specificity of the proteins obtained in example 1, example 3 and comparative examples 1 to 3 to the mycoplasma pneumoniae antibody were determined by the following method:

the above-mentioned 96-well ELISA plates were coated with 12 proteins at a concentration of 100ng/ml and incubated overnight at 4 ℃. PBST wash 1 time; blocking with 1% BSA in PBS, 100. Mu.l/well, incubating at 37 ℃ for 4h, discarding the solution, and mixing the above 101 sera from children with pneumonia to be tested with PBST: 100 dilution, 100. Mu.l/well ELISA plate, 37 ℃ incubation for 2H, discard solution, PBST wash 5 times, addition of horseradish peroxidase-labeled goat anti-human IgM (1 dilution), 100. Mu.l/well, 37 ℃ incubation for l H, discard solution, PBST wash 5 times, addition of o-phenylenediamine (o-PD) substrate solution 100. Mu.l/well, color development for 30min, addition of 0.2mM H ₂ SO ₄ The reaction was stopped, 50. Mu.l/well, A450 and A630 were measured with a microplate reader, the cut-off value was determined by statistical analysis, and a value greater than the cut-off value was selected as positive, the results are shown in Table 1.

TABLE 1 detection results of sensitivity and specificity of the proteins obtained in example 1, example 3, and comparative examples 1 to 3 to Mycoplasma pneumoniae antibodies

P30B1

P30 fragment

1

P30 fragment 2

P30 fragment 3

P30 fragment 4

Sensitivity of the probe

86.00％

82.00％

74.00％

72.00％

66.00％

Specificity of

96.08％

92.16％

74.51％

76.47％

In addition, the invention also refers to the experimental example to detect the sensitivity and specificity of the protein P30 to the Mycoplasma pneumoniae antibody, and the results are 70.00% and 78.43%, so that the P30B1 fragment provided in the example 1 and the P30 fragment 1 provided in the example 3 of the invention have better sensitivity and specificity compared with the P30 and other fragments, and have development prospects.

Example 5

The embodiment provides a preparation method of a fusion protein P1 fragment-linker-P30B 1, which comprises the following specific steps:

adopting the P1 fragment-PET 28a recombinant plasmid preserved in the laboratory as a template, designing a primer, amplifying by adopting a PCR method to obtain a P1 fragment gene product containing a homologous arm with the N tail end of P30B1, as shown in SEQ ID No.15, cutting, purifying and recycling gel, and determining the nucleic acid concentration to be 61 ng/mu l; using P30B1-pET32a recombinant plasmid as a template, designing a primer, amplifying by adopting a PCR method to obtain a P30B1 gene product containing a homologous arm with the C terminal of the P1 fragment, and determining the concentration of nucleic acid to be 58 ng/mu l; determining pET28a plasmid with the nucleic acid concentration of 152 ng/microliter, performing double enzyme digestion by using BamH1 and Xho1, and performing temperature control at 37 ℃ for 30min to obtain a linearized vector plasmid; cloning the P1 fragment gene product and the P30B1 gene product to pET28a, 4 mu l of the P1 fragment gene product and 4 mu l of the P30B1 gene product by adopting LQ connection and using a seamless cloning technology, 2 mu l of a linearized pET28a vector and 10 mu l of 2 × ultra clone express mix (purchased from Nanjing Nuo Touzin Biotechnology GmbH), 37 ℃ and 30min; adding 10 mu l of seamless clone ligation product into 100 mu l of self-made Top10 competent cells, carrying out ice bath for 30min, carrying out hot shock for 88s at 42 ℃ in water bath, then placing the seamless clone ligation product into ice water for 2-5min, adding 700 mu l of nonreactive LB liquid culture medium, carrying out shaking culture at 37 ℃ and 250rpm for 1h, centrifuging (4500 rpm,5 min), discarding 600 mu l of supernatant, carrying out heavy suspension on the cells, taking 100 mu l of bacterial liquid, coating the bacterial liquid on LB solid culture medium containing 100 mu g/ml ampicillin, placing the dried bacterial liquid in a constant temperature incubator upside down, carrying out overnight culture at 37 ℃ for 14-16h, picking 3 monoclonal transformants the transformants on the next day, respectively inoculating the transformants in 4ml of LB liquid culture medium containing 100 mu g/ml ampicillin, carrying out shaking culture at 37 ℃ and 250rpm for 6-8h, and sequencing to verify whether the recombinant vector construction is correct.

Then, extracting the correctly sequenced bacterial liquid, measuring the concentration to be 182 ng/mul, taking 1 mul to add into 100 mul self-made Rosetta (DE 3) competent cells, ice-bathing for 30min, then hot-shocking for 88s at 42 ℃ in water bath, putting into ice water for 2-5min, adding 700 mul non-resistant LB liquid culture medium, shaking and culturing for 1h at 37 ℃ and 250rpm, centrifuging (4500rpm, 5 min), discarding 600 mul supernatant, re-suspending the thalli, taking 100 mul LB solid culture medium coated on the bacterial liquid containing 100 mul g/ml ampicillin and 50 mul g/ml chloramphenicol, pouring the dried bacterial liquid into a constant temperature incubator, culturing overnight at 37 ℃ (14-16 h), next day, picking 3 single clones, respectively inoculating into 15ml LB liquid culture medium containing 100 mul g/ml ampicillin and 50 mul g/ml chloramphenicol, shaking and culturing for 4h at 37 ℃ and 250rpm, adding IPTG,25 ℃ to obtain the final concentration, 250rpm, then centrifuging and collecting supernatant, discarding supernatant, centrifuging and inducing and culturing for 4h.

The steps of ultrasonic bacteria breaking and protein purification are the same as those in example 1, and the electrophoresis result of the fusion protein P1 fragment-LQ-P30B 1 is shown in FIG. 10 f.

Experimental example 2

In order to prove the clinical applicability of the protein provided by the invention, 101 serum samples of clinical pneumonia children confirmed by a commercial kit (SERODIA-MYCO II) are collected in the experimental example, and the detection result is 50 positive parts and 51 negative parts.

Then, using mycoplasma pneumoniae holophage antigen, the TrxA-6 × His-S-tag-P30B1 recombinant protein provided in example 1, the S-tag-P30B1 protein obtained in example 1, the MBP-6 × His-P30B1 protein obtained in example 4, the S-tag-P30B1-6 × His protein obtained in example 2, the TrxA-6 × His-tag-P30 fragment 1 protein obtained in example 3, the P1 fragment-LQ-P30B 1 fusion protein obtained in example 5, the TrxA-6 × His-S tag-P30 fragment 2 protein obtained in comparative example 1, the TrxA-6 × His-S tag-P30 fragment 3 protein obtained in comparative example 2, the TrxA-6 × His-S tag-P30 fragment 4 protein obtained in comparative example 3, the P1 fragment provided in example 5, and the 30 fragments provided in example 1, the statistical detection method and the negative detection method were performed, and the detection results were provided in the following manner:

the above-mentioned 12 proteins were used to coat 96-well ELISA plates at a concentration of 100ng/ml, and incubated overnight at 4 ℃. PBST wash 1 time; blocking with 1% BSA in PBS, 100. Mu.l/well, incubating at 37 ℃ for 4h, discarding the solution, and mixing the above 101 sera from children with pneumonia to be tested with PBST: 100 dilution, 100. Mu.l/well ELISA plate, 37 ℃ incubation for 2H, discard solution, PBST wash 5 times, addition of horseradish peroxidase-labeled goat anti-human IgM (1 dilution), 100. Mu.l/well, 37 ℃ incubation for l H, discard solution, PBST wash 5 times, addition of o-phenylenediamine (o-PD) substrate solution 100. Mu.l/well, color development for 30min, addition of 0.2mM H ₂ SO ₄ Termination of the reaction, 50Mu.l/hole, measuring A450 and A630 by using a microplate reader, determining cut-off value by adopting statistical analysis, selecting the value larger than cut-off as positive, and detecting results are shown in tables 2-13.

TABLE 2 Whole cell antigen Performance assays

TABLE 3TrxA-6 × his-S-tag-P30B1 protein Performance test

TABLE 4 detection of S-tag-P30B1 protein Performance

TABLE 5 MBP-6 × his-P30B1 protein Performance assays

TABLE 6 detection of the Performance of the fusion protein S-tag-P30B1+6 XHis protein

TABLE 7 detection of the Performance of the fusion protein P1 fragment-LQ-P30B 1 protein

TABLE 8 protein P1 fragment protein Performance assays

TABLE 9 P30 protein Performance assays

TABLE 10 TrxA-6 XHis-Stag-P30 fragment 1 Performance test

TABLE 11 TrxA-6 XHis-Stag-P30 fragment 2 Performance assays

TABLE 12 TrxA-6 XHis-S-tag-P30 fragment 3 Performance test

TABLE 13 TrxA-6 XHis-S-tag-P30 fragment 4 Performance test

TABLE 14 sensitivity, specificity, false positive, false negative detection of Mycoplasma pneumoniae holosomatic antigen, respective proteins to Mycoplasma pneumoniae antibody

The results of the experiment were analyzed as follows:

1. the mycoplasma pneumoniae holosomatic antigen is commercially available, and the P1 fragment protein and the P30 protein are self-made according to the P1 fragment and the P30 fragment in the laboratory. The sensitivity, specificity, false positive, and false negative of mycoplasma pneumoniae holoantigen and each protein to mycoplasma pneumoniae antibodies are shown in table 14.

The sensitivity and specificity of the P30B1 fusion protein containing the protein tag are superior to those of a whole bacterial antigen and a P30 protein, the false positive and false negative probabilities of the protein P30B on the detection of mycoplasma pneumoniae are greatly lower, the sensitivity and specificity of a fusion protein P1 fragment-linker-P30B 1 are respectively 94.00 percent and 98.04 percent, and the sensitivity and specificity are superior to those of the P30B1 fusion protein containing the protein tag and the P1 fragment protein.

Because the label S-tag is not combined and identified with the mycoplasma pneumoniae antibody, the sensitivity and specificity of the MBP-6 XHis-P30B 1 protein are lower than those of S-tag-P30B1, probably because the molecular weight of the protein label MBP is large, the combination of an antigenic determinant and the antibody is hindered on a spatial structure, and the functions of the S-tag-P30B1 protein and the P30B1 protein are similar, so that the sensitivity and specificity of the P30B1 protein to the mycoplasma pneumoniae antibody are better than those of the mycoplasma pneumoniae holoantigen and the P30 protein, and the false negative and false positive are lower than those of the mycoplasma pneumoniae holosomatic antigen and the P30 protein.

Therefore, the sensitivity and specificity of the P30B1 protein, trxA-6 xHis-S-tag-P30B 1 protein, MBP-6 xHis-P30B 1 protein, S-tag-P30B1-6 xHis protein, fusion protein P1 fragment-LQ-P30B 1 and P1 fragment protein to the mycoplasma pneumoniae antibody are presumed to be superior to those of the mycoplasma pneumoniae holoantigen and the P30 protein, and the false negative and false positive are lower than those of the mycoplasma pneumoniae holoantigen and the P30 protein.

2. The protein sequence similarity of the P30 fragment 1 and the P30B1 is 88.70%, the structure has higher similarity, therefore, the TrxA-6 xHis-S-tag-P30B 1 protein has the same function as the TrxAA-6 xHis-S tag-P30 fragment 1, the detection result shows that the sensitivity and the specificity of the TrxA-6 xHis-S tag-P30B1 protein to the mycoplasma pneumoniae antibody are similar in numerical value, and the TrxA-6 xHis-S tag-P30B1 protein and the Mycoplasma pneumoniae antibody can be used as independent antigens for preparing a mycoplasma pneumoniae diagnostic reagent; the sensitivity and specificity of the TrxA-6 xHis-S-tag-P30B 1 protein to mycoplasma pneumoniae antibodies are superior to those of the TrxA-6 xHis-tag-P30 fragment 2, the TrxA-6 xHis-tag-P30 fragment 3 and the TrxA-6 xHis-S tag-P30 fragment 4, and the antigenicity of the P30B1 fragment is superior to that of the P30 fragment 2, the P30 fragment 3 and the P30 fragment 4 (the homology of the P30 fragment 4 and the P30B1 is 71.4%). It is inferred that the sequence having 88% or more homology with P30B1 has the effect of enhancing sensitivity and specificity as compared with P30.

3. The sensitivity and specificity of the TrxA-6 xHis-S-tag-P30B 1 protein, the S-tag-P30B1 protein and the MBP-6 xHis-P30B 1 recombinant fusion protein to the mycoplasma pneumoniae antibody are superior to those of a whole bacterial antigen and a P30 protein, and the fusion protein can be used as a candidate component of a novel mycoplasma pneumoniae detection kit antigen.

4. The amino acid structure similarity of the S-tag-P30B1 protein and the fusion protein S-tag-P30B1-6 XHis is 96.31 percent, and the highly similar structure determines that the fusion protein has similar functions, so the application effect is almost the same.

5. The TrxA-6 xHis-S-tag-P30B 1 protein is different from the fusion tag of the S-tag-P30B1 protein and the MBP-6 xHis-P30B 1 protein, wherein the fusion tag MBP has a large molecular weight, and the spatial structure prevents the combination of an antigenic determinant and an antibody; compared with the S-tag, the TrxA-6 xHis-S-tag has better stability to the N-terminal structure of the P30B1, so that the downstream application effect of the TrxA-6 xHis-S-tag-P30B 1 protein is better.

6. The fusion protein P1 segment-linker-P30B 1 fuses the P1 segment and the P30B1, the two segments are antigenic determinants of main adhesion factors of mycoplasma pneumoniae, the detection sensitivity can be improved after the two segments are fused, and the joint use of recombinant proteins with different antigenic determinants is further proved, so that the detection sensitivity can be improved, and the defect that the single antigen is weak in sensitivity is overcome.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. The first protein is characterized in that the N end to the C end of the first protein consists of a TrxA label, a first connecting peptide, a6 xHis label, a second connecting peptide, an S-tag label, a third connecting peptide and a first protein fragment which are sequentially connected, wherein the amino acid sequence of the first protein fragment is shown in SEQ ID No. 1; the amino acid sequence of the first connecting peptide is shown as SEQ ID No.5, the amino acid sequence of the second connecting peptide is shown as SEQ ID No.8, and the amino acid sequence of the third connecting peptide is shown as SEQ ID No. 6.

2. The second protein is characterized by consisting of an MBP label, a fourth connecting peptide, a6 XHis label, a fifth connecting peptide and a first protein fragment which are sequentially connected from the N end to the C end, wherein the amino acid sequence of the first protein fragment is shown in SEQ ID No. 1; the amino acid sequence of the fourth connecting peptide is shown in SEQ ID No.7, and the fifth connecting peptide is GS.

3. The third protein is characterized by consisting of an S-tag label, a sixth connecting peptide, a first protein fragment, a seventh connecting peptide and a6 XHis label which are sequentially connected from the N end to the C end, wherein the amino acid sequence of the first protein fragment is shown in SEQ ID No.1, the amino acid sequence of the sixth connecting peptide is shown in SEQ ID No.6, and the seventh connecting peptide is LE.

4. The fourth protein is characterized by comprising an S-tag label, a multiple cloning site and a first protein fragment which are sequentially connected from the N end to the C end, wherein the amino acid sequence of the first protein fragment is shown as SEQ ID No.1, and the amino acid sequence of the multiple cloning site is shown as SEQ ID No. 6.

5. A nucleic acid molecule encoding a protein according to any one of claims 1 to 4.

6. A biomaterial comprising the nucleic acid molecule of claim 5, wherein the biomaterial comprises any one of (a) to (d):

(a) An expression cassette;

(b) A recombinant vector;

(c) Recombinant eukaryotic cells;

(d) Recombinant prokaryotic cells.

7. The biomaterial according to claim 6, characterized in that the recombinant vector selects plasmid pET32a as vector.

8. The biological material according to claim 6, wherein the recombinant prokaryotic cell is E.coli as host cell.

9. The biomaterial of claim 8, wherein the host cell is e.

10. A method for producing a protein according to any one of claims 1 to 4, which comprises expressing and translating the nucleic acid molecule of claim 5 or the biomaterial according to any one of claims 6 to 9 to obtain a protein product, purifying and concentrating the protein product to obtain the protein.

11. Use of the protein according to any one of claims 1 to 4, the nucleic acid molecule according to claim 5, the biomaterial according to any one of claims 6 to 9, or the protein produced by the production method according to claim 10, for the production of a pneumonia vaccine product or a mycoplasma pneumoniae test product.

12. A reagent for detecting Mycoplasma pneumoniae, comprising the protein according to any one of claims 1 to 4, the protein encoded by the nucleic acid molecule according to claim 5, the protein expressed from the biomaterial according to any one of claims 6 to 9, or the protein produced by the production method according to claim 10.

13. A method for the detection of Mycoplasma pneumoniae which is not diagnostic or therapeutic using the Mycoplasma pneumoniae detection reagent according to claim 12, wherein the Mycoplasma pneumoniae detection method comprises chemiluminescence, ELISA, colloidal gold rapid detection, agar diffusion, agglutination, or immunoblotting.