CN112979769A

CN112979769A - Amino acid sequence, protein, preparation method and application thereof

Info

Publication number: CN112979769A
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: 2021-06-18
Anticipated expiration: 2041-03-08
Also published as: CN112979769B

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 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 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 MP cell membrane, and comprises 275 amino acid residues, the relative molecular mass is about 29.7Kd, and the whole length of the gene sequence is 825 bp. Studies have shown that the P30 protein can stimulate host immune response, and is another important adhesin and immunogen besides the adhesion protein P1. The P30 antigen was 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 pneumonia [ J ] in Vaccine Immunol.2008,15(2):215 and 220.), but the P30 antigen used a wild-type gene, which is low in expression level, and thus, the application in diagnosis and Vaccine development is limited. The adhesion protein P30 is adhered to the cell membrane, and part of the polypeptide sequence is embedded into the hydrophobic region of the membrane, so that the fat 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 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 encoded 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 biological material constructed by using the nucleic acid molecule, thereby achieving efficient expression of the above protein.

The fifth object of the present invention is to provide a method for producing the protein, whereby the protein can be obtained.

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 as described in the previous embodiments.

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 1-3, 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, a 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 polyclonal site as shown in SEQ ID No. 8.

Preferably, the protein consists of an MBP label, a6 XHis label 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 label and the 6 XHis label, a fifth connecting peptide is connected between the 6 XHis label 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 label, a first protein fragment and a6 XHis label which are sequentially connected from an N end to a C end, a sixth connecting peptide is connected between the S-tag label and the first protein fragment, a seventh connecting peptide is connected between the first protein fragment and the 6 XHis label, 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 embodiment, 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 foregoing 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 provided by the invention has no 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 a mycoplasma pneumoniae holobacterium.

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 prediction result of the transmembrane region of P30;

FIG. 6 shows the prediction result of the transmembrane region of P30B 1;

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 pattern of purified TrxA-6 XHis-Stag-P30B 1 by Q-HP ion exchange chromatography in example 1; c is an SDS-PAGE picture of a sample after the TrxA-6 xHis-Stag-P30B 1 protein is purified and concentrated in example 1; d is an SDS-PAGE picture of the TrxA-6 xHis-Stag-P30B 1 protein in example 1 after restriction enzyme digestion by Thrombin;

in FIG. 10, a is an SDS-PAGE pattern of the purified S-tag-P30B1 protein in example 1; b is the SDS-PAGE picture of the purified S-tag-P30B1-6 XHis protein in example 2; c is the SDS-PAGE picture of the MBP-6 x his-P30B1 protein purified by the Ni ion affinity chromatographic column in example 4; d is the SDS-PAGE pattern of the concentrated sample after the protein MBP-6 XHis-P30B 1 desalinization 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 sample of the 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, 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 is also to be understood that, unless otherwise expressly specified or limited, the term "coupled" is used broadly, and may, for example, refer to directly coupled devices or indirectly coupled devices 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 of the sequences according to the 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 the invention having more than 88% homology with the amino acid sequence of SEQ ID NO.1, the following are exemplified:

for example one

The homology analysis between 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 numbers of the SEQ ID NO.11 sequence and the SEQ ID NO.9 are 521, the full length of the SEQ ID NO.11 sequence is 579, the similarity is 88.67% (525/579), the nucleic acid sequence has high identity, 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 rows of sequences to be compared, the upper row is the SEQ ID NO.9 sequence, and the lower row is the SEQ ID NO.11 sequence.

The number of the same 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 homology between the two sequences of SEQ ID NO.1 and SEQ ID NO.10 can be inferred, the amino acid sequence comparison result is shown in FIG. 2, wherein the upper row of the two sequences for comparison 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 between 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 same number of bases of the sequence SEQ ID NO.13 as the sequence SEQ ID NO.9 is 267, the full length of the sequence SEQ ID NO.13 is 600 bases, and the similarity of the sequence SEQ ID NO.13 to the sequence 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 present invention, a protein which is generated by performing a fusion experiment on the fragment P30B1 and has a specific recognition function of more than 88% on Mycoplasma pneumoniae antibodies still falls into the protection scope of the present 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 a 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:

the P30 protein has a fat-soluble transmembrane region fragment, and is analyzed by bioinformatics software, as shown in fig. 5, 70 amino acids (shown as B in fig. 5) at the N-terminal 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 specificity of P30 cannot meet the application requirements.

The invention firstly selects 5 fragments from P30 antigen: the fragment P30B1 (amino acid residues: 98-274), the P30 fragment 1 (amino acid residue: 118-274), the P30 fragment 2 (amino acid residue: 107-161), the P30 fragment 3 (amino acid residues: 98-204) and the P30 fragment 4 (amino acid residues: 28-204) are respectively constructed on a pET32a vector, an N-terminal fusion tag TrxA +6 xHis + S-tag is fused, and the detection is carried out by an ELisa method after expression and purification, and the result shows (Table 14) that the sensitivity and the specificity of TrxA +6 xHis + S-tag-P30B1 are obviously higher than those of other four fragments and P30 protein. Fragment 1 of P30 is a shortened sequence of P30B1, having 88.4% homology to the P30B1 protein, and it is inferred that the sequence having 88% or more homology to P30B1 has the effect of enhancing sensitivity and specificity as compared to P30.

The P30B1 protein protected by the invention is a periplasmic space expression protein derived after removing the transmembrane region of the P30 protein, the protein has no transmembrane region, as shown in FIG. 6, the protein directly exists in the periplasmic space after expression, 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 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 antigen can be developed into independent antigen 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, protective modification is carried out at least at the N terminal of the protein P30B1, optional protective modification comprises connection of protective protein, and optional protective protein comprises more than one of S-tag, TrxA tag, GST tag, MBP tag, SUMO tag or NusA tag, or more than one of 6 xHis tag, S-tag, TrxA tag, GST tag, MBP tag, SUMO tag or NusA tag.

However, it should be noted that protein P30B1 was rapidly degraded and extremely unstable when the N-terminus of the P30B1 sequence was linked to only 6 × His, indicating that N-terminal linkage to only 6 × His did not help maintain the N-terminal structural integrity of the protein.

In the embodiment of the invention, TrxA-6 XHis-S-tag-P30B 1 protein, S-tag-P30B1 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 protein and mycoplasma pneumoniae holosomatic antigen (commercially available) is tested, and the result shows (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 false positive and false negative probabilities of the protein P30B on the detection of mycoplasma pneumoniae are greatly lower, the sensitivity and specificity of the fusion protein P1 fragment-linker-P30B 1 are 94.00 percent and 98.04 percent respectively, and the sensitivity and specificity are superior to those of the fusion protein containing the protein tag P30B1 and the protein containing the P1 fragment.

Some embodiments of the invention are described in detail below with reference to the accompanying drawings. The embodiments described below and the features of the embodiments 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 adhesion protein P30 of mycoplasma pneumoniae (FH strain) by using ProtScale software, wherein the amino acid sequence is 275 amino acids 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 lipid 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, an amino acid label is 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 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), SD _ like (GGRGGT), and the optimized gene fragment SEQNO.9 is obtained by adopting a total synthesis gene method, is constructed between the multiple cloning sites BamH1 and Xho1 of the Pet32a vector, and is entrusted to Nanjing Kingsry Biotech limited company for synthesis to obtain the recombinant vector P30B1-pET32 a.

1.2 construction of recombinant plasmid MBP-P30B1-pET28a

Carrying out double 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 digestion for subsequent experiments, carrying out agarose gel electrophoresis on the digestion product of the P30B1-pET32a plasmid obtained in the step 1, carrying out gel cutting purification, recovering a target gene fragment with the size of about 500bp, and determining the nucleic acid concentration to be 65 ng/mu l by using a nanodrop instrument.

And (3) 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 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 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 and adding the solution into 100 mu l of escherichia coli E.coli Rosetta (DE3) 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.

1.4 cells were collected by centrifugation, and 8mL of lysine buffer (20mM PBS +500mM NaCl, pH7.4) 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 that the ultrasonic power is 400W, the ultrasonic is 4s, the ultrasonic is stopped for 3s, and the circulation is carried out 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; and respectively reserving 20 mu l of supernatant and sediment, performing SDS-PAGE, and screening recombinant bacteria for soluble expression of the target protein by a small-scale expression test.

1.6 pretreatment of Ni ion affinity chromatography column: 5 column volumes were washed with buffer B (20mM PBS, 1M imidazole, pH7.4) 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, named FT, was collected in a clean vial and 20. mu.l was retained.

1.8, cleaning: after the flow-through was completed, the column was washed with lyss buffer (20mM PBS +500mM NaCl, pH7.4) until the baseline was stabilized (about 10 column volumes) and the flow rate was unchanged.

1.9 elution: the eluates were collected and 20. mu.l of the eluates were retained by using imidazole concentrations of 20mM, 40mM, 80mM, 160mM, 250mM, and 1M, respectively, in this order.

1.10 the samples retained in the above steps are subjected to SDS-PAGE in the order of adding 5. mu.l of 5Xloading buffer to the EP tubes of all the retained samples in the order of elution, and the results are 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, pH7.4), 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, pH7.4) 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. The 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 enzyme-cleaved sample was purified by Ni ion affinity chromatography column, the operation was 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 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 steps as 10-13 to obtain purified S-tag-P30B1 protein, wherein as shown in a figure 10a, the S-tag-P30B1 protein is proved to be still capable of keeping the P30B1 protein sequence complete, and the N end of P30B1 is not degraded, so that the 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 an 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 primers, amplifying by using a PCR method to obtain an S-tag-P30B1-6 XHis gene fragment, cutting gel, purifying and recycling, determining the nucleic acid concentration to be 73 ng/microliter, constructing on a linearized vector pET28a by using a seamless cloning technology, producing 3 microliter of the S-tag-P30B1-6 XHis gene product, 2 microliter of the linearized pET28a vector, 5 microliter of 2 × ultra clone express Jingmix (purchased from Nannunop Biotech GmbH), 37 ℃ and 30 min; adding 10 mu l of seamless cloning ligation product into 100 mu l of self-made Top10 competent cells, carrying out ice bath for 30min, carrying out heat shock for 88s at 42 ℃ in water bath, then placing the seamless cloning 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 (4500rpm, 5min), discarding 600 mu l of supernatant, carrying out heavy suspension on the bacteria, taking 100 mu l of bacterial liquid, coating the bacterial liquid on LB solid culture medium containing 100 mu g/ml ampicillin, placing the bacterial liquid in a constant temperature incubator after drying, carrying out overnight culture at 37 ℃ (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 verifying whether the recombinant vector construction is correct by bacterial liquid sequencing.

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

Extracting plasmid from bacterial liquid with correct sequencing, measuring the concentration to be 164 ng/mu l, adding 1 mu l into 100 mu l of self-made Rosetta (DE3) 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 mu l of non-antibiotic LB liquid culture medium, carrying out shaking culture at 37 ℃ and 250rpm for 1h, centrifuging (4500rpm and 5min), discarding 600 mu l of supernatant, re-suspending the thalli, taking 100 mu l of bacterial liquid, coating the bacterial liquid on an LB solid culture medium containing 100 mu g/ml ampicillin and 50 mu g/ml chloramphenicol, pouring the dried bacterial liquid into a constant temperature incubator, carrying out overnight culture at 37 ℃ (14-16h), carrying out secondary transformation on the next day, picking 3 monoclonals, respectively inoculating into 15ml LB liquid culture medium containing 100 mu g/ml ampicillin and 50 mu g/ml chloramphenicol, carrying out shaking culture at 37 ℃ and 250rpm for 4h, adding 1mM IPTG, inducing expression at 25 deg.C and 250rpm, culturing for 4 hr, centrifuging (4500rpm, 5min), collecting thallus, and discarding supernatant.

The procedure of disrupting the bacteria with ultrasound and purifying the protein was 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 was substituted for the 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:

construction of a MBP-6 XHis-P30B 1-pET28a recombinant plasmid in which MBP is linked to 6 XHis via the enzyme-cleavage site protection base FEGS and TEV enzyme-cleavage site ENLYFQS in that order, and 6 XHis and P30B1 are linked via the enzyme-cleavage site BamH1, and then the recombinant plasmid was transformed and plated on LB 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 column and Q-HP ion exchange column to obtain MBP-6 XHis-P30B 1 protein, and the results are 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 was taken and added with 600. mu.g of TEV enzyme, the enzyme digestion was carried out overnight at 4 ℃, 20. mu.l of the digested sample was added with 5. mu.l of loading buffer, and SDS-PAGE was carried out, and the result is shown in FIG. 10e, and the result in FIG. 10e shows that only protein bands of MBP and TEV enzyme and no protein band of 6 XHis-P30B 1 exist in the sample after TEV digestion, 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 to the N-terminal.

Comparative example 1

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

Experimental example 1

The sensitivity and specificity of the proteins obtained in the above examples 1, 3 and 1 to 3 to the mycoplasma pneumoniae antibody are detected by the following detection method:

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 PBS containing 1% BSA, incubating at 37 ℃ for 4h at 100. mu.l/well, discarding the solution, and mixing the above 101 sera for children with pneumonia to be tested with PBST in l: diluting with 100, adding enzyme-labeled plate at 100 μ l/well, incubating at 37 deg.C for 2H, discarding solution, washing with PBST for 5 times, adding horse radish peroxidase-labeled goat anti-human IgM (1:2000 dilution), incubating at 100 μ l/well, incubating at 37 deg.C for l H, discarding solution, washing with PBST for 5 times, adding o-phenylenediamine (o-PD) substrate solution at 100 μ l/well, developing for 30min, adding 0.2mM H₂SO₄Stopping reaction, 50 mul/hole, measuring A450 and A630 by enzyme-labeling instrument, determining cut-off value by statistical analysis, and selecting the value greater than cut-off as positiveThe 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%, respectively, therefore, the P30B1 fragment provided in the example 1 and the P30 fragment 1 provided in the example 3 of the invention both show better sensitivity and specificity compared with the P30 and other fragments, and have development prospect.

Example 5

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

the P1 fragment-PET 28a recombinant plasmid preserved in the laboratory is used as a template, a primer is designed, a P1 fragment gene product containing a homology arm with the N tail end of P30B1 is obtained by PCR amplification, as shown in SEQ ID No.15, gel is cut, purified and recovered, and the concentration of the measured nucleic acid is 61 ng/mul; 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 homology arm with the C terminal of the P1 fragment, and determining the nucleic acid concentration to be 58 ng/mu l; determining pET28a plasmid with nucleic acid concentration of 152 ng/microliter, performing double enzyme digestion with BamH1 and Xho1 at 37 deg.C for 30min to obtain linearized vector plasmid; cloning the P1 fragment gene product and the P30B1 gene product to pET28a, 4 mu l of P1 fragment gene product, 4 mu l of P30B1 gene product, 2 mu l of linearized pET28a vector, 10 mu l of 2 xultra clone express mix (purchased from Nanjing Nozao Touzan Biotech Co., Ltd.) at 37 ℃ for 30min by adopting LQ connection and a seamless cloning technology; 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 heat shock at 42 ℃ for 88s, then putting 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 (4500rpm, 5min), 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, pouring the bacterial liquid into a constant temperature incubator after the bacterial liquid is dried, carrying out overnight culture at 37 ℃ (14-16h), picking 3 monoclonal transformants the transformants, 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 verifying whether the recombinant vector construction is correct by sequencing of the.

Then, extracting the bacterial liquid with correct sequencing, measuring the concentration to be 182 ng/mul, adding 1 mul into 100 mul of self-made Rosetta (DE3) 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 non-antibiotic LB liquid culture medium, carrying out shaking culture at 37 ℃ and 250rpm for 1h, centrifuging (4500rpm and 5min), discarding 600 mul of supernatant, re-suspending the thalli, taking 100 mul of bacterial liquid, coating the 100 mul of bacterial liquid on an LB solid culture medium containing 100 mul/ml ampicillin and 50 mul/ml chloramphenicol, pouring the bacterial liquid into a constant temperature incubator after drying, carrying out overnight culture at 37 ℃ (14-16h), picking 3 monoclonals the next day, respectively inoculating into 15ml of LB liquid culture medium containing 100 mul/ml ampicillin and 50 mul g/ml chloramphenicol, 37 ℃, after shaking culture at 250rpm for 4h, 1mM IPTG was added to the cells at 25 ℃ and induced expression at 250rpm for 4h, and the cells were collected by centrifugation (4500rpm, 5min) and the supernatant was discarded.

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 children with clinical pneumonia 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 holosomatic antigen, TrxA-6 XHis-S-tag-P30B 1 recombinant protein provided in example 1, S-tag-P30B1 protein obtained in example 1, MBP-6 XHis-P30B 1 protein obtained in example 4, S-tag-P30B1-6 XHis protein obtained in example 2, TrxA-6 XHis-Stag-P30 fragment 1 protein obtained in example 3, P1 fragment-LQ-P30B 1 fusion protein obtained in example 5, protein TrxA-6 XHis-S tag-P30 fragment 2 obtained in comparative example 1, protein TrxA-6 XHis-S tag-P30 fragment 3 obtained in comparative example 2, protein TrxA-6 XHis-S tag-P30 fragment 3 protein obtained in comparative example 3, protein TrxA-6 XHis-S tag-P30 fragment provided in example 5, and the P30 fragment provided in example 5, detecting 101 serum samples according to the following detection method, and counting positive results and negative results, wherein the detection method comprises the following steps:

respectively adopting the above-mentioned 12 protein-coated 96-well ELISA plates, coating concentration is 100ng/ml, and incubating at 4 deg.COvernight. PBST wash 1 time; blocking with PBS containing 1% BSA, incubating at 37 ℃ for 4h at 100. mu.l/well, discarding the solution, and mixing the above 101 sera for children with pneumonia to be tested with PBST in l: diluting with 100, adding enzyme-labeled plate at 100 μ l/well, incubating at 37 deg.C for 2H, discarding solution, washing with PBST for 5 times, adding horse radish peroxidase-labeled goat anti-human IgM (1:2000 dilution), incubating at 100 μ l/well, incubating at 37 deg.C for l H, discarding solution, washing with PBST for 5 times, adding o-phenylenediamine (o-PD) substrate solution at 100 μ l/well, developing for 30min, adding 0.2mM H₂SO₄The reaction was stopped, 50. mu.l/well, A450 and A630 were measured with a microplate reader, cut-off was determined by statistical analysis, a value greater than cut-off was selected as positive, and the test results are shown in tables 2 to 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 test

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 detection of P30 protein Performance

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

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

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 by the laboratory according to the P1 fragment and the P30 fragment. 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 the fusion protein P1 fragment-linker-P30B 1 are 94.00 percent and 98.04 percent respectively, and the sensitivity and specificity are superior to those of the fusion protein containing the protein tag P30B1 and the protein containing the P1 fragment.

Because the tag 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 tag MBP is large, the binding of an antigenic determinant and the antibody is prevented in a spatial structure, 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 holomycoplasmal antigen and the P30 protein, and the false negative and false positive are lower than those of the mycoplasma pneumoniae holomycoplasmal antigen and the P30 protein.

Therefore, the sensitivity and specificity of the protein P30B1, TrxA-6 XHis-S-tag-P30B 1, protein S-tag-P30B1, protein MBP-6 XHis-P30B 1, protein S-tag-P30B1-6 XHis, fusion protein P1 fragment-LQ-P30B 1 and protein P1 fragment to the mycoplasma pneumoniae antibody are better than those of the mycoplasma pneumoniae holosomatic antigen and the protein P30, and the false negative and false positive are lower than those of the mycoplasma pneumoniae holosomatic antigen and the protein P30.

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 TrxAA-6 xHis-S tag-P30 fragment 1 are similar, and the TrxAA-6 xHis-S tag-P30 fragment and the TrxAA-6 xHis-S tag-P3526 fragment 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 antibody are better than 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, which shows that the antigenicity of the P30B1 fragment is better than that of the P30 fragment 2, the P30 fragment 3 and the P30 fragment 4 (the homology of the P30 fragment 4 to the P30B1 is 71.4%). It is concluded that the sequences having 88% or more homology to P30B1 have the effect of enhancing sensitivity and specificity compared to 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 three recombinant fusion proteins to the mycoplasma pneumoniae antibody are superior to those of a whole thallus 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%, and the similar structure determines that the protein has similar functions, so the application effect is almost the same.

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

6. The fusion protein P1 fragment-linker-P30B 1 fuses a P1 fragment and a P30B1 fragment, the two fragments are antigenic determinants of main adhesion factors of mycoplasma pneumoniae, the detection sensitivity can be improved after the two fragments are fused, and the combined 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 made up.

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 the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Zhuhaili medical diagnostic products Co., Ltd

<120> amino acid sequence, protein, preparation method and application thereof

<160> 15

<170> PatentIn version 3.5

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Pro Ile Val Lys Arg Lys Glu Lys Arg Leu Leu Glu Glu Lys Glu Arg

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35 40 45

Gln Gln Ala Leu Glu Gln Gln Ala Ala Ala Glu Ala His Ala Glu Ala

50 55 60

Glu Val Glu Pro Ala Pro Gln Pro Val Pro Val Pro Pro Gln Pro Gln

65 70 75 80

Val Gln Ile Asn Phe Gly Pro Arg Thr Gly Phe Pro Pro Gln Pro Gly

85 90 95

Met Ala Pro Arg Pro Gly Met Pro Pro His Pro Gly Met Ala Pro Arg

100 105 110

Pro Gly Phe Pro Pro Gln Pro Gly Met Ala Pro Arg Pro Gly Met Pro

115 120 125

Pro His Pro Gly Met Ala Pro Arg Pro Gly Phe Pro Pro Gln Pro Gly

130 135 140

Met Ala Pro Arg Pro Gly Met Pro Pro His Pro Gly Met Ala Pro Arg

145 150 155 160

Pro Gly Phe Pro Pro Gln Pro Gly Met Ala Pro Arg Pro Gly Met Gln

165 170 175

Pro Pro Arg Pro Gly Met Pro Pro Gln Pro Gly Phe Pro Pro Lys Arg

180 185 190

<210> 11

<211> 579

<212> DNA

<213> Artificial sequence

<400> 11

ggctttagcg cgctggcgat cattctgggt ctggcgatcg gcctgccgat tgtgaagcgt 60

aaagaaaagc gtctgctgga ggaaaaagaa cgtcaggagc aactggcgga gcagctgcaa 120

cgtatcagcg cgcagcaaga ggaacagcaa gcgctggaac aacaggcggc ggcggaggcg 180

catgcggagg cggaagtgga gccggcgccg caaccggtgc cggttccgcc gcagccgcaa 240

gttcagatta actttggtcc gcgtaccggt tttccgccgc agccgggtat ggcgccgcgt 300

ccgggtatgc cgccgcaccc gggcatggcg ccgcgtccgg gcttcccgcc gcaacctggt 360

atggcgccgc gtcctggcat gccgccgcac cccggcatgg cgccgcgtcc tggttttccg 420

ccgcagcctg gcatggcgcc gcgtcccggc atgccgccgc acccaggcat ggcgccgcgt 480

ccaggcttcc cgccgcaacc aggtatggcg ccgcgtcccg gtatgcaacc gccgcgtccg 540

ggcatgccgc cgcagccggg cttcccgccg aagcgttaa 579

<210> 12

<211> 200

<212> PRT

<213> Artificial sequence

<400> 12

Asp Gly Asn Thr Ser Ser Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr

1 5 10 15

Gly Asn Asp Val Val Gly Val Gly Arg Leu Ser Glu Ser Asn Ala Ala

20 25 30

Lys Met Asn Asp Asp Val Asp Gly Ile Val Arg Thr Pro Leu Ala Glu

35 40 45

Leu Leu Asp Gly Glu Gly Gln Thr Ala Asp Thr Gly Pro Gln Ser Val

50 55 60

Lys Phe Lys Ser Pro Asp Gln Ile Asp Phe Asn Arg Leu Phe Thr His

65 70 75 80

Pro Val Thr Asp Leu Phe Asp Pro Val Thr Met Leu Val Tyr Asp Gln

85 90 95

Tyr Ile Pro Leu Phe Ile Asp Ile Pro Ala Ser Val Asn Pro Lys Met

100 105 110

Val Arg Leu Lys Val Leu Ser Phe Asp Thr Asn Glu Gln Ser Leu Gly

115 120 125

Leu Arg Leu Glu Phe Phe Lys Pro Asp Gln Asp Thr Gln Pro Asn Asn

130 135 140

Asn Val Gln Val Asn Pro Asn Asn Gly Asp Phe Leu Pro Leu Leu Thr

145 150 155 160

Ala Ser Ser Gln Gly Pro Gln Thr Leu Gln Pro Ile Val Lys Arg Lys

165 170 175

Glu Lys Arg Leu Leu Glu Glu Lys Glu Arg Gln Glu Gln Leu Ala Glu

180 185 190

Gln Leu Gln Arg Ile Ser Ala Gln

195 200

<210> 13

<211> 603

<212> DNA

<213> Artificial sequence

<400> 13

gatggtaaca ccagcagcac caacaacctg gcgccgaaca ccaacaccgg caacgacgtg 60

gttggtgtgg gccgtctgag cgaaagcaac gcggcgaaaa tgaacgatga cgtggacggt 120

atcgttcgta ccccgctggc ggagctgctg gatggcgagg gtcagaccgc ggacaccggt 180

ccgcagagcg tgaagtttaa aagcccggat caaatcgact tcaaccgtct gtttacccac 240

ccggttaccg acctgttcga cccggtgacc atgctggttt acgatcagta tattccgctg 300

tttatcgaca ttccggcgag cgttaacccg aagatggtgc gtctgaaagt tctgagcttc 360

gataccaacg agcaaagcct gggtctgcgt ctggagttct tcaaaccgga tcaagacacc 420

cagccgaaca acaacgtgca ggttaacccg aacaacggtg actttctgcc gctgctgacc 480

gcgagcagcc aaggtccgca gaccctgcag ccgattgtga agcgtaaaga aaagcgtctg 540

ctggaggaaa aagaacgtca ggagcaactg gcggagcagc tgcaacgtat cagcgcgcag 600

taa 603

<210> 14

<211> 70

<212> PRT

<213> Artificial sequence

<400> 14

Ala Thr Leu Ile Leu Val Gln His Asn Asn Thr Glu Leu Thr Glu Val

1 5 10 15

Lys Ser Glu Leu Ser Pro Leu Asn Val Val Leu His Ala Glu Glu Asp

20 25 30

Thr Val Gln Ile Gln Gly Lys Pro Ile Thr Glu Gln Ala Trp Phe Ile

35 40 45

Pro Thr Val Ala Gly Cys Phe Gly Phe Ser Ala Leu Ala Ile Ile Leu

50 55 60

Gly Leu Ala Ile Gly Leu

65 70

<210> 15

<211> 178

<212> PRT

<213> Artificial sequence

<400> 15

Trp Leu Val Gly Gln Leu Pro Ser Thr Ser Asp Gly Asn Thr Ser Ser

1 5 10 15

Thr Asn Asn Leu Ala Pro Asn Thr Asn Thr Gly Asn Asp Val Val Gly

20 25 30

Val Gly Arg Leu Ser Glu Ser Asn Ala Ala Lys Met Asn Asp Asp Val

35 40 45

Asp Gly Ile Val Arg Thr Pro Leu Ala Glu Leu Leu Asp Gly Glu Gly

50 55 60

Gln Thr Ala Asp Thr Gly Pro Gln Ser Val Lys Phe Lys Ser Pro Asp

65 70 75 80

Gln Ile Asp Phe Asn Arg Leu Phe Thr His Pro Val Thr Asp Leu Phe

85 90 95

Asp Pro Val Thr Met Leu Val Tyr Asp Gln Tyr Ile Pro Leu Phe Ile

100 105 110

Asp Ile Pro Ala Ser Val Asn Pro Lys Met Val Arg Leu Lys Val Leu

115 120 125

Ser Phe Asp Thr Asn Glu Gln Ser Leu Gly Leu Arg Leu Glu Phe Phe

130 135 140

Lys Pro Asp Gln Asp Thr Gln Pro Asn Asn Asn Val Gln Val Asn Pro

145 150 155 160

Asn Asn Gly Asp Phe Leu Pro Leu Leu Thr Ala Ser Ser Gln Gly Pro

165 170 175

Gln Thr

Claims

1. An amino acid sequence, wherein said amino acid sequence comprises any one of:

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

2. The amino acid sequence of claim 1, wherein the amino acid sequence is as set forth in SEQ ID No. 3.

3. A polypeptide comprising an amino acid sequence according to claim 1 or claim 2.

4. A protein comprising the amino acid sequence of claim 1 or claim 2.

5. The protein of claim 4, wherein the protein comprises a first protein fragment encoded by the amino acid sequence of claim 1, 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 N-terminus and the C-terminus of the first protein fragment are linked to a protective protein;

6. The protein according to claim 5, wherein 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 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;

7. The protein of claim 6, wherein 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, a 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.

8. The protein of any one of claims 5 to 7, wherein 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 as SEQ ID No.5, the amino acid sequence of the second connecting peptide comprises a thrombomin enzyme cutting site and an enzyme cutting site protecting amino acid residue, and is shown as SEQ ID No.8, and the amino acid sequence of the third connecting peptide comprises a polyclonal site;

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

9. A nucleic acid molecule for editing a protein according to any one of claims 4 to 8, comprising a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID No. 1;

10. A biomaterial comprising a nucleic acid molecule according to claim 9, 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;

preferably, the recombinant vector uses a plasmid pET32a as a vector;

11. A method for producing a protein according to any one of claims 4 to 8, which comprises expressing and translating the nucleic acid molecule according to claim 9 or the biological material according to claim 10 to obtain a protein product, purifying and concentrating the protein product to obtain the protein.

12. Use of the protein according to any one of claims 4 to 8, the nucleic acid molecule according to claim 9, the biomaterial according to claim 10 or the protein produced by the production method according to claim 11 for the production of a pneumonia vaccine product or a mycoplasma pneumoniae test product.

13. A reagent for detecting Mycoplasma pneumoniae, comprising the protein according to any one of claims 4 to 8, the protein encoded by the nucleic acid molecule according to claim 9, the protein expressed from the biomaterial according to claim 10, or the protein produced by the production method according to claim 11.

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