CN101665533A - Protein active polypeptide, coding gene thereof, expression vector thereof, expression fungus thereof and application - Google Patents

Abstract

The invention relates to a protein active polypeptide, a coding gene thereof, an expression vector thereof, expression fungus thereof, and an application thereof. The protein active polypeptide is: (a) the polypeptide formed by head-to-tail serial connection of 2 to 12 polypeptide ZYZ22 or homologous sequences thereof; (b) the derived polypeptide formed by the polypeptide in step (a) through the substitution and/or loss and/or adding of one or a plurality of amino acid residues; and (c) the polypeptide containing any polypeptide in steps (a) and (b) and having functions of protein stability and active protection. The DNA sequence is: (a) the DNA sequence coding the protein active polypeptide; and (b) the DNA sequence crossed with the DNA sequence in (a) under strict conditions. The recombinant expression vector contains any DNA sequence of the invention. The expression fungus contains any recombinant expression vector or any DNA sequence of the invention. The polypeptide can maintain stability and bioactivity of the protein preparation, and can be applied to protein stability and bioactivity protective agents.

Description

Protein active polypeptide and its coding gene, expression vector, expression bacteria and application

technical field

The invention relates to biotechnology, in particular to a protein stabilization and biological activity protection polypeptide and its coding gene, recombinant expression vector, expression bacteria and the application of the protein stabilization and biological activity protection polypeptide.

Background technique

In the fields of modern biological science, chemistry and medicine, various protein preparations with biological activity are widely used. Protein preparations are generally stored in two forms: liquid and solid (dry powder). The protein solution in the first way is easily denatured at room temperature, reducing or losing its biological activity, so it needs to be stored in a low-temperature environment, but during storage and transportation, it may be separated from the low-temperature environment due to various reasons, causing protein Denatured precipitation and decreased biological activity. The second way is to prepare the protein into dry powder and store it at room temperature, but the dehydration process caused by freezing and drying in the freeze-drying process can cause damage and denaturation of the protein, resulting in a decrease in biological activity. In addition, during the use of protein solution preparations, repeated freezing and thawing can significantly reduce the biological activity of proteins. In order to maintain the stability of the protein and protect the biological activity of the protein, various protective agents are often added to the protein preparation, for example: serum protein and glycerin and other protective agents are added to the protease solution, and the serum protein can prevent protease from occurring due to hydrophobic interactions. Glycerol can avoid protease aggregation caused by hydrophobic interaction and protect the biological activity of protease. Various protective agents such as sugars, polyols, serum proteins, surfactants and amino acids are added to the protein freeze-dried preparations, which can form hydrogen bonds with the polar groups on the protein surface to prevent the protein from losing water during the freeze-drying process Afterwards, hydrogen bonds are directly exposed to the surrounding environment, reducing protein damage, denaturation, and aggregation.

Contents of the invention

The first object of the present invention is to provide a protein stabilization and biological activity protection polypeptide.

The second object of the present invention is to provide a gene encoding the protein stabilization and biological activity protection polypeptide of the present invention.

The third object of the present invention is to provide a recombinant expression vector of the protein stabilization and biological activity protection polypeptide of the present invention.

The fourth object of the present invention is to provide an expression bacterium expressing the protein stabilization and biological activity protection polypeptide of the present invention.

The fifth object of the present invention is to provide the application of the protein stabilization and biological activity protection polypeptide of the present invention.

The inventors noticed that a highly conserved 22-amino acid sequence and its homologous sequences (homologous sequences generally having 1-9 amino acid substitutions) are mostly distributed in plant embryo proteins. The 22 amino acid sequence and its homologous sequences are mainly composed of hydrophilic amino acids. The inventor speculates that the 22 amino acid sequence can prevent dehydration caused by high temperature, drying or freezing, and damage and denaturation of the protein caused by repeated freezing and thawing, maintain the stability of the protein and protect the biological activity of the protein. The inventor named this 22 amino acid sequence "polypeptide ZYZ22", and expressed and obtained a polypeptide containing 2-12 polypeptide ZYZ22 or its homologous sequence through genetic engineering methods, and added it to protein liquid preparations and protein solid preparations, It can effectively improve the stability of the protein at room temperature, prevent the denaturation and inactivation of the protein during the freeze-drying process, prevent the inactivation of the protein during repeated freezing and thawing, and protect the biological activity of the protein.

The protein stabilization and biological activity protection polypeptide of the present invention is the polypeptide described in (a) or (b) or (c) as follows:

(a) A polypeptide composed of 2-12 polypeptides ZYZ22 or its homologous sequences connected in series from end to end, and the polypeptides ZYZ22 are connected by 0-5 amino acids; the polypeptide ZYZ22 is described in SEQ ID NO.1 in the sequence listing The amino acid sequence of , its homologous sequence is a sequence with 1-9 amino acid substitutions;

(b) The polypeptide described in (a) is a polypeptide derived from (a) that has protein stabilization and activity protection functions through the substitution and/or deletion and/or addition of one or several amino acid residues;

(c) A polypeptide containing the polypeptide described in (a) or (b) and having protein stabilization and activity protection functions.

The amino acid sequence of the polypeptide ZYZ22 is as follows: VNKMGEYKDYAAEKAKEGKDAT.

The protein stabilization and biological activity protection specifically can prevent protein damage, denaturation and biological activity loss caused by high temperature, drying, freezing and repeated freezing and thawing.

In order to facilitate the purification of the inventive protein stabilization and biological activity protection polypeptide, the present invention may have a Poly-His tag or a GST tag attached to the amino-terminal or carboxyl-terminal of the polypeptide described in (a) or (b) or (c).

The protein stabilization and biological activity protection polypeptide of the present invention is preferably the amino acid sequence described in SEQ ID NO.2 in the sequence table, which is a polypeptide composed of two homologous sequences of polypeptide ZYZ22 in series. Named as ZYZ22-2 polypeptide; better is the amino acid sequence described in SEQ ID NO.3 in the sequence table, which is a polypeptide composed of six homologous sequences of polypeptide ZYZ22 in series, and the applicant named it ZYZ22 -6 polypeptide; better is the amino acid sequence described in SEQ ID NO.4 in the sequence table, which is a polypeptide formed by tandeming the homologous sequences of 12 polypeptides ZYZ22, and the applicant named it ZYZ22-12 polypeptide .

The polypeptide described in (c) in the present invention is preferably the ZYZ-L3 polypeptide described in SEQ ID NO.5 in the sequence listing.

The protein stabilization and biological activity protection polypeptide of the present invention can be artificially synthesized, or its coding gene can be firstly synthesized and then biologically expressed. Wherein (b) the coding gene of the polypeptide can be obtained by deleting the codon of one or several amino acid residues in the coding gene of (a) the polypeptide, and/or performing a missense mutation of one or several base pairs , and/or obtained by connecting the coding sequence of Poly-His tag or GST tag at its 5' end.

The DNA sequence of the present invention is the DNA sequence described in (a) or (b) below:

(a) DNA sequence encoding any protein stabilization and biological activity protection polypeptide of the present invention;

(b) a DNA sequence that can hybridize with the DNA sequence described in (a) under stringent conditions; the stringent conditions are in 6×SSC, 0.5% SDS solution, hybridization at 65° C., and then using 2×SSC, Wash the membrane once with 0.1% SDS, 1×SSC, and 0.1% SDS.

The nucleotide sequence described in SEQ ID NO.6 in the sequence listing is the nucleotide sequence encoding the ZYZ22-2 polypeptide. The nucleotide sequence described in SEQ ID NO.7 in the sequence listing is the nucleotide sequence encoding the ZYZ22-6 polypeptide described in SEQ ID NO.3 in the sequence listing. The nucleotide sequence described in SEQ ID NO.8 in the sequence listing is the nucleotide sequence encoding the ZYZ22-12 polypeptide described in SEQ ID NO.4 in the sequence listing. The nucleotide sequence described in SEQ ID NO.9 in the sequence listing is the nucleotide sequence encoding the ZYZ-L3 polypeptide described in SEQ ID NO.5 in the sequence listing.

The recombinant expression vector of the present invention contains any one of the above-mentioned DNA sequences of the present invention. Recombinant expression vectors can be constructed using existing prokaryotic expression vectors.

The expression bacterium of the present invention contains any recombinant expression vector or any DNA sequence of the present invention.

The protein stabilization and biological activity protection polypeptide expression and purification method of the present invention is to transform the recombinant expression vector into expression bacteria such as Escherichia coli, induce expression, collect the bacteria, cell disruption, centrifugation, affinity chromatography purification, and gel filtration. Finally, protein stabilization and biological activity protection peptides are obtained.

The protein stabilization and biological activity protection polypeptide of the present invention can be applied to protein stabilization and protein activity protection agents.

Experiments have proved that the protein stabilization and biological activity protection polypeptide of the present invention, as a protein stabilization and protein activity protection agent, can prevent or reduce protein denaturation, aggregation and precipitation caused by high temperature, freezing, drying or repeated freezing and thawing after being added to the protein solution. It can prevent or reduce the loss of protein biological activity caused by high temperature, freezing, drying or repeated freezing and thawing. For example: after the ZYZ22-2 polypeptide described in SEQ ID NO.2 in the sequence table is added to the lactate dehydrogenase solution, it is frozen with liquid nitrogen, and then thawed at 25°C. The resulting decrease in enzyme activity has an obvious protective effect on the protein biological activity during repeated freezing and thawing. The ZYZ22-2 polypeptide described in SEQ ID NO.2 in the sequence listing and trehalose are added to the lactate dehydrogenase solution, and under high temperature (such as 63°C) conditions, they can synergize with trehalose to protect the activity of lactate dehydrogenase. The effect is higher than that of ZYZ22-2 polypeptide or trehalose alone.

The protein stabilization and biological activity protection polypeptide of the present invention is used as a protein stabilization and activity protection agent, which can maintain the stability of the protein in liquid form under high temperature treatment or repeated freezing and thawing treatment, protect the biological activity of the protein, and have great advantages in the application of protein preparations. Significance.

Description of drawings

Figure 1 is the physical map of the prokaryotic expression vector pET-ZYZ22-2.

Fig. 2 is the electrophoresis diagram of the purified ZYZ22-2 polypeptide.

Fig. 3 is a graph showing the protective effect of ZYZ22-2 polypeptide on the activity of lactate dehydrogenase (LDH) under repeated freezing and thawing conditions.

Fig. 4 is a graph showing the synergistic protective effect of ZYZ22-2 polypeptide and trehalose on the activity of lactate dehydrogenase (LDH) under high temperature conditions.

Fig. 5 is a graph showing the protective effects of ZYZ-22-6, ZYZ-22-12, and ZYZ-L3 polypeptides on lactate dehydrogenase (LDH) activity under repeated freezing and thawing conditions.

Detailed ways

The experimental methods in the following examples are conventional methods unless otherwise specified.

Example 1: Construction of prokaryotic expression vector and recombinant strain of polypeptide ZYZ22-2

Total RNA was extracted from the immature seeds of soybean Bainong No. 6 (provided by Baicheng Agricultural Science Research Institute of Jilin Province), and the RNA was synthesized into cDNA by reverse transcriptase. Primers were designed according to the soybean late embryonic development abundant protein 3 histone (LEA3) sequence (the sequence number of Genebank is M80664) retrieved in the Genebank database as follows:

5'-ATGGCGTCCAAGAAACAAGA-3'

5'-TGCGTCTATATATACTAATA-3'.

The cDNA obtained by reverse transcription was used as a template for PCR amplification, and the PCR product was detected by 0.8% agarose gel electrophoresis, and a band with a molecular weight of about 1400bp was obtained, which was consistent with the expected result. This fragment was recovered using an agarose gel recovery kit (Takara). The recovered fragment was connected with pGEM-T Easy (purchased from Promega Company), and the connection product was transformed into E. coli Top10 competent cells with reference to the method of Cohen et al. (Proc Natl Acad Sci, 69: 2110). Positive clones were screened with the carbenicillin resistance marker on the marker, and recombinant plasmids containing recovered fragments were obtained. The T7 and SP6 promoter sequences on the recombinant plasmid vector were used as primers to determine its nucleotide sequence, and the sequencing results showed that the amplified soybean late embryonic development abundant protein 3 histone (LEA3) gene was composed of 1389 deoxyribose nucleus The recombinant plasmid containing the nucleotide sequence was named pGEM-LEA3.

The pGEM-LEA3 plasmid containing the LEA3 gene was digested with EcoR I and Not I, and connected to the pET-28a(+) vector that had undergone the same double digestion to obtain a recombinant vector, named ZYZ-L3. Transform into Escherichia coli BL21 Star to obtain BL21-ZYZ3 strain.

Primers were designed according to the nucleotide sequence of the ZYZ22-2 polypeptide in the late-stage abundant protein 3 histone of soybean embryonic development, i.e. the nucleotide sequence described in SEQ ID NO.6 in the sequence listing:

5'-GAATTCGGTTCCAAGGTCGGAGAG-3'

5'-AAGCTTTTAGGTCGTCTTCTTCGCTTC-3'.

Using pGEM-LEA3 as a template, PCR amplification was carried out, and the PCR product was detected by 0.8% agarose gel electrophoresis, and a band with a molecular weight of about 140bp was obtained, which was consistent with the expected result. This fragment was recovered using an agarose gel recovery kit (Takara). The recovered fragment was connected to the pET-28a (+) vector, and the insertion sites were EcoR I and HindIII. With reference to the method of Cohen et al. (Proc Natl Acad Sci, 69: 2110), the connection product was transformed into Escherichia coli Top10 competent cells, Positive clones were screened according to the kanamycin resistance marker on the pET-28a(+) vector to obtain recombinant plasmids containing recovered fragments. The T7 promoter sequence on the recombinant plasmid vector was used as a primer to determine its nucleotide sequence, and the sequencing results showed that it was the nucleotide sequence encoding the ZYZ22-2 polypeptide in the soybean late embryonic development abundant protein group 3 gene (LEA3), Its open reading frame (ORF) is the 1st to 132nd deoxyribonucleotides from the 5' end of SEQ ID NO.6 in the sequence listing, and the encoded amino acid sequence is the 1st to 44th amino acid of SEQ ID NO.2 in the sequence listing .

The above-mentioned recombinant plasmid vector containing the deoxyribonucleotide sequence described in SEQ ID NO.6 in the sequence listing is named pET-ZYZ22-2, see accompanying drawing 1.

The recombinant vector pET-ZYZ22-2 was transformed into Escherichia coli BL21 STAR to obtain the recombinant strain BL21/ZYZ22-2.

Similarly, design the following primers to perform PCR amplification using the ZYZ-L3 plasmid as a template:

5'-CACCGAATTCTCTAGAGGTTCCAAGGTCGGAGAGTAC-3'

5'-GTCCAAGCTTCCTAGCACTAGTCCCCAACGTCGTATCTTT-3'.

Repeat the above process of agarose gel electrophoresis and PCR product recovery to obtain the nucleotide sequence encoding the ZYZ22-6 polypeptide, and clone it into the pET-28a(+) vector, and the obtained plasmid is named pET-ZYZ22-6. Its open reading frame (ORF) is the 1st to 429th deoxyribonucleotides from the 5' end of SEQ ID NO.7 in the sequence listing, and the encoded amino acid sequence is the 1st to 143rd of SEQ ID NO.3 in the sequence listing amino acid.

Insert the nucleotide sequence encoding the ZYZ22-6 polypeptide into the recombinant vector pET-ZYZ22-6 constructed above again, the fragment restriction site is Nhe I and HindIII, the vector restriction site is Spe I and HindIII, and the obtained The recombinant vector expressing ZYZ22-12 polypeptide is named pET-ZYZ22-12. Its open reading frame (ORF) is the deoxyribonucleotide at positions 1 to 864 from the 5' end of SEQ ID NO.8 in the sequence listing, and the encoded amino acid sequence is the polypeptide of SEQ ID NO.4 in the sequence listing, namely ZYZ22 -12 polypeptides.

Example 2: Expression of polypeptide ZYZ22-2

Inoculate BL21/ZYZ22-2 single clone in a small amount of culture medium, culture at 37°C until the absorbance value (OD ₆₀₀ ) is 0.8, add isopropyl-BD thiogalactoside (IPTG) to 0.2mM, and induce at 37°C After 4 hours, the cells were collected by centrifugation at 4000g, resuspended in 2×sample buffer, treated in a water bath at 100°C for 5 minutes, centrifuged at 10000g for 10 minutes, the supernatant was taken for 12% SDS-PAGE, stained, and the results were observed. Compared with the experimental control group BL21/pET-28a, a specific protein band appeared in BL21/ZYZ22-2, which was close to the expected molecular weight of ZYZ22-2 polypeptide of 10kDa.

Example 3: Purification of polypeptide ZYZ20-2

Inoculate BL21/ZYZ22-2 monoclonal into a small amount of culture medium, culture overnight at 37°C; transfer to 1000ml LB liquid medium at a ratio of 1:100, culture at 37°C until the absorbance value (OD ₆₀₀ ) is 0.8, add isopropyl -BD thiogalactoside (IPTG) to 0.2mM, induced at 37°C for 4h, centrifuged at 4000g to collect the bacteria, resuspended in PBS, sonicated in an ice bath, centrifuged at 20000g at 4°C for 10 minutes, collected the supernatant, and referred to metal Instructions for use of chelating affinity chromatography medium, use Chelating Sepharose Fast Flow column to purify the fusion protein, and use Superdex 30 PG prepacked column to perform gel filtration on the obtained preliminary purified protein, collect the target protein, and perform 12% SDS-PAGE. The results showed that there was a specific band at the expected molecular weight, while the rest of the protein bands basically disappeared, and a relatively high-purity ZYZ22-2 polypeptide was obtained, as shown in Figure 2.

Example 4: Protective effect of ZYZ22-2 polypeptide on lactate dehydrogenase (LDH) activity under repeated freezing and thawing conditions

In the lactate dehydrogenase reaction system, the concentration of lactate dehydrogenase is 62.5ng/ml. ZYZ22-2 polypeptide and lactate dehydrogenase in different concentrations were mixed according to the mass ratio of 1:10, 1:5, 1:1, 5:1, 1:10 respectively, and then divided into two groups. One set was used to directly measure the activity of lactate dehydrogenase. The other group was frozen in liquid ammonia for 1 minute, completely thawed on a constant temperature mixer at 25°C, and treated 10 times repeatedly. Protection under repeated freezing and thawing. The same amount of serum protein BSA was added as the control group.

The results showed that after repeated freezing and thawing, the activity of lactate dehydrogenase without adding protective agent almost completely disappeared, and the enzyme activity was only about 1% of that before treatment. After adding the ZYZ22-2 polypeptide with a mass concentration ratio of 1:1, the residual enzyme activity of lactate dehydrogenase increased to 11% of that before treatment. Adding an equal proportion of BSA to the control group can increase the residual enzyme activity of lactate dehydrogenase, but it is significantly lower than that of the ZYZ22-2 polypeptide, see Figure 3. Experiments show that the ZYZ22-2 polypeptide has a good protective effect on the activity of lactate dehydrogenase under repeated freezing and thawing conditions.

Example 5: Synergistic protective effect of ZYZ22-2 polypeptide and trehalose on lactate dehydrogenase (LDH) activity under high temperature conditions

Different amounts of ZYZ22-2 polypeptide were mixed with 62.5 ng/ml lactate dehydrogenase. The mass ratios of ZYZ22-2 polypeptide and lactate dehydrogenase after mixing are 0:1, 1:5, 1:1 and 5:1 respectively. Divide the mixture into two groups. One set was used for the direct measurement of lactate dehydrogenase activity. The other group was kept on a constant temperature mixer at 63°C for 5 minutes, and the activity of lactate dehydrogenase was measured, and the activity of lactate dehydrogenase before and after adding ZYZ22-2 polypeptide was compared, and the effect of ZYZ22-2 polypeptide on lactate dehydrogenase at high temperature was analyzed. protective effect. The same amount of serum protein BSA was added as the control group.

At the same time, trehalose with a final concentration of 250 ng/ml was added to the above experimental system, and after the same process as above, the activity of lactate dehydrogenase was measured.

The results showed that after the lactate dehydrogenase was treated at high temperature, the activity of the lactate dehydrogenase without adding protective agent was only 16%. When the added ZYZ22-2 polypeptide and BSA have the same quality, the lactate dehydrogenase activity is relatively close. After adding 50mM trehalose to the experimental system, when adding the same mass of ZYZ22-2 polypeptide and BSA, the LDH enzyme activity was significantly higher than adding ZYZ22-2 polypeptide and BSA alone. Moreover, when the mass ratio of ZYZ22-2 polypeptide, BSA protein and LDH enzyme is 1:5, 1:1 and 5:1, the lactate dehydrogenase activity of ZYZ22-2 polypeptide is higher than that of BSA, see Figure 4. The experimental results show that the ZYZ22-2 polypeptide has a certain protective effect on the activity of lactate dehydrogenase at high temperature, and in the case of simultaneous presence of trehalose, it can exert a synergistic effect to improve the protective effect on the activity of lactate dehydrogenase.

The ZYZ22-12 polypeptide was also subjected to repeated freeze-thaw treatment experiments. The experimental results showed that when the mass ratio of ZYZ22-12 polypeptide to LDH enzyme was 1:1, after 10 repeated freeze-thaw treatments, the residual activity of LDH enzyme was higher than that of ZYZ22- 2 polypeptide was increased by more than 1 times, indicating that ZYZ22-12 polypeptide has a stronger protective effect on LDH enzyme activity than ZYZ22-2 polypeptide, see Figure 5. According to the experimental results and theoretical speculation, other series of 22 amino acid series polypeptides have protective effects on LDH enzymes, and with the increase of the number of 22 amino acid series series, the protective effect on LDH enzymes can be reduced under the condition of adding the same quality series of polypeptides. The effect is also improved accordingly. Similarly, for a polypeptide containing 22 amino acid sequences, the ZYZ-L3 polypeptide as described in SEQ ID NO.5 has also been subjected to repeated freeze-thaw treatment experiments. The experimental results showed that when the mass ratio of ZYZ-L3 polypeptide to LDH enzyme was 1:1, after 10 repeated freeze-thaw treatments, the residual activity of LDH enzyme was significantly higher than that of the control group added with BSA. According to the experimental results and theoretical speculation, other polypeptides containing 22 amino acid sequences all have protective effects on LDH enzymes. See attached drawing 5.

sequence listing

<110> Shenzhen University

<120>Protein active polypeptide and its coding gene, expression vector, expression bacteria and application

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<170>PatentIn version 3.1

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atggcgtcca agaaacaaga ggagcgagct gaggcagctg cgaaagttgc tgccaaagaa 60

ctcgaacaag tcaacagaga aagaagagac cgtgatttcg gtgttgttgc tgaacaacaa 120

caacaacatc atcaggaaga tcaacaaaaa cgtggtgtaa tcgggtccat gtttaaggcg 180

gtgcaagaca cctacgagaa cgccaaggaa gctgtcgttg gcaagaaaga agctactaat 240

aacgcgtaca gtaatacaga ggttattcac gatgttaaca ttcagcccga tgacgtgtcg 300

gcaacggggg aagtaaggga catatcagcc acaaagactc atgatatcta cgattctgcc 360

acggacaaca acaacaacaa aaccggttcc aaggtcggag agtacgcaga ttacgcttct 420

cagaaggcca aggaaacaaa agatgcaacg atggaaaaag ctggagagta cacagattat 480

gcttcgcaga aagcgaagga agcgaagaag acgaccatgg agaagggtgg agaatacaag 540

gattactctg cggagaaagc taaggagaga aaagatgcta ctgtgaataa gatgggagag 600

tataaggact atgctgcgga gaaagccaaa gaggggaaag atgctactgt gaataaaatg 660

ggagagtata aggactatgc tgcggagaaa acgaaagagg ggaaagatgc cactgtgaat 720

aagatggggag agtataagga ttacactgcg gagaaggcga aagagggga agatacgacg 780

ttggggaagc ttggggagct gaaggacacg gcttcggatg cggcgaagag ggccgtgggt 840

tacttgagcg gcaagaaaga ggaaactaaa gagatggctt cggagaccgc cgaggcgacg 900

gcgaataagg caggggagat gaaggaggca acaaagaaaa agacggcgga gaccgcggag 960

gcggcgaaga ataaggcggg ggagatcaag gacagagccg cggagacggc ggaggccgcg 1020

aaaaacaaga ccgcggagac cgcggaagtg acgaagaata aggctttgga gatgaaggat 1080

gcagcgaagg acaggaccgc tgagacaacg gatgcggcga agcagaaaac tgcacaggca 1140

aaggagaaca ccaaggaaaa tgtgagtggt gcaggtgaaa ctgcaaggag gaaaatggaa 1200

gagccaaagc ttcaaggtaa agaagggtat gggggccgtg gagacaaggt ggtggtgaaa 1260

gtggaagaga gtcgaccagg ggcaattgcg gaaacgctga aagccgccga ccagattgcg 1320

ggacagacct tcaacgatgt aggacgcttc gatgaagagg gtgtcgtcaa tgtggagcgc 1380

cgcaagaaa 1389

Claims (10)

1. A protein stabilization and biological activity protection polypeptide, which is the polypeptide described in (a) or (b) or (c) as follows: (a) A polypeptide composed of 2-12 polypeptides ZYZ22 or its homologous sequences connected in series from end to end, and the polypeptides ZYZ22 are connected by 0-5 amino acids; the polypeptide ZYZ22 is described in SEQ ID NO.1 in the sequence listing The amino acid sequence of , its homologous sequence is a sequence with 1-9 amino acid substitutions; (b) The polypeptide described in (a) is a polypeptide derived from (a) that has protein stabilization and activity protection functions through the substitution and/or deletion and/or addition of one or several amino acid residues; (c) A polypeptide containing the polypeptide described in (a) or (b) and having protein stabilization and activity protection functions. 2. The protein stabilization and biological activity protection polypeptide according to claim 1, characterized in that: it is the amino acid sequence described in SEQ ID NO.2 in the sequence listing. 3. The protein stabilization and biological activity protection polypeptide according to claim 1, characterized in that: it is the amino acid sequence described in SEQ ID NO.3 in the sequence listing. 4. The protein stabilization and biological activity protection polypeptide according to claim 1, characterized in that: it is the amino acid sequence described in SEQ ID NO.4 in the sequence listing. 5. The protein stabilization and biological activity protection polypeptide according to claim 1, characterized in that: it is the amino acid sequence described in SEQ ID NO.5 in the sequence listing. 6. A DNA sequence, which is a DNA sequence as described in (a) or (b) below: (a) the DNA sequence encoding the protein stabilization and biological activity protection polypeptide described in claim 1; (b) a DNA sequence that can hybridize with the DNA sequence described in (a) under stringent conditions; the stringent conditions are in 6×SSC, 0.5% SDS solution, hybridization at 65° C., and then using 2×SSC, Wash the membrane once with 0.1% SDS, 1×SSC, and 0.1% SDS. 7. The DNA sequence according to claim 6, characterized in that: it is the nucleotide sequence described in SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8 or SEQ ID NO.9 in the sequence listing . 8. A recombinant expression vector comprising any DNA sequence according to claim 6. 9. An expression bacterium comprising any one of the recombinant expression vectors as claimed in claim 8 or any one of the DNA sequences as claimed in claim 6. 10. The application of the protein stabilizing and biologically active protecting polypeptide according to claim 1 in a protein stabilizing and biologically active protecting agent.

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