Optimized Lv-Bip-like protein and application thereof in improving heat resistance of malate dehydrogenase

Abstract

The invention provides an optimized Lv-Bip-like protein and an application of the optimized Lv-Bip-like protein in improving the heat resistance of malate dehydrogenase. The method comprises the following steps: cloning an Lv-Bip-like gene from a litopenaeus vannamei body, optimizing the Lv-Bip-like gene, further constructing a recombinant vector by using a gene cloning technology, carrying out induced expression of prokaryotic protein, purifying the prokaryotic protein by using an affinity chromatography method, and carrying out ultrafiltration concentration to obtain the optimized Lv-Bip-like protein. The MDH protein is unstable when encountering heat, and experiments prove that after the optimized rBip-like protein is added under the heating condition, aggregation of the MDH protein can be reduced by optimizing the Lv-Bip-like protein, and then the heat stability of the MDH protein is improved. Therefore, the optimized Lv-Bip-like protein can be applied to feed additives, especially formula improvement of pig feed, and has a good market application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to an optimized Lv-Bip-like protein and an application thereof in improving the heat resistance of malate dehydrogenase. Background Art

[0002] Malate dehydrogenase (MDH) is mainly involved in metabolic pathways such as TCA cycle, photosynthesis, and C4 cycle. In most prokaryotes and eukaryotes, such as pig heart MDH, MDH forms homodimers with a molecular weight between 32 and 37 kDa. In thermophilic bacteria, MDH has high thermal stability, but in most organisms, MDH has poor thermal stability, which affects its function.

[0003] Studies have shown that alcohols and the addition of disulfide bonds or hydrogen bonds can improve the thermal stability of MDH. MDH protein is unstable when exposed to heat, and improving the thermal stability of MDH plays an important role in ensuring the function of malate dehydrogenase. Summary of the invention

[0004] In view of the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide an optimized Lv-Bip-like protein and its application in improving the heat resistance of malate dehydrogenase.

[0005] In order to solve the above technical problems, the technical solution to be adopted in the present invention is: The present invention provides an optimized Lv-Bip-like protein, whose amino acid sequence is shown in SEQ ID NO.4.

[0006] Furthermore, it is obtained by changing the amino acid at position 224 from aspartic acid to glutamine and the amino acid at position 368 from proline to cysteine ​​based on the amino acid sequence shown in SEQ ID NO.3.

[0007] The present invention also provides a gene encoding the Lv-Bip-like protein, and its nucleotide sequence is shown in SEQ ID NO.5.

[0008] The present invention also provides a Lv - Bip-like A recombinant expression vector of a gene, Lv - Bip-like The nucleotide sequence of the gene is shown in SEQ ID NO.5.

[0009] The recombinant expression vector is a pET-32 a (+) vector.

[0010] The present invention also provides application of the Lv-Bip-like protein in improving the heat resistance of malate dehydrogenase.

[0011] Furthermore, the Lv-Bip-like protein improves the heat resistance of malate dehydrogenase by inhibiting its thermal aggregation.

[0012] Furthermore, the malate dehydrogenase is derived from pig heart.

[0013] The present invention also provides the use of the Lv-Bip-like protein in the preparation of an animal feed additive for improving the heat resistance of the protein.

[0014] Furthermore, the animals are pigs or broilers.

[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: the present invention clones Lv - Bip-like Gene, after optimization, further use gene cloning technology to construct a recombinant vector and perform induced expression of prokaryotic protein, purify the prokaryotic protein by affinity chromatography, and concentrate by ultrafiltration. The malate dehydrogenase (MDH) used in the present invention is derived from pig heart, has thermal instability, and is easily decomposed by heat. Experiments have shown that after adding the optimized Lv-Bip-like protein under heating conditions, it is proved that the protein can reduce the aggregation of MDH protein, thereby improving the thermal stability of malate dehydrogenase. Therefore, the optimized Lv-Bip-like protein can be used in feed additives, especially in the formulation improvement of pig feed, and has good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 for Lv -rBip-like gene recombinant vector double enzyme digestion verification experiment results; Lane 1: original plasmid; Lane 2: recombinant plasmid double enzyme digestion verification; M: DNA marker; Figure 2 for Lv -rBip-like induced expression and in vitro affinity purification results; where M: protein marker; lanes 1-2: before and after induction; lanes 3-4: supernatant and precipitate; Figure 3 is the protein standard curve; Figure 4 The results of MDH in vitro thermal aggregation experiment. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with embodiments. The following embodiments are for explanation of the present invention but the present invention is not limited to the following embodiments. Example 1

[0018] 1. Sequence transformation The present invention screened out the transcriptome database of Penaeus vannamei Lv - Bip-like Gene (Endoplasmicreticulum chaperone BiP-like), the sequence is shown in SEQ ID NO: 1, and the ORF region sequence is shown in SEQ ID NO. 2 (1968bp). The Bip like gene belongs to the HSP 70 protein family, plays an important biological function in stabilizing endoplasmic reticulum stress in organisms, and can also alleviate the stress caused by misfolded proteins. Lv - Bip-like The amino acid sequence of the protein translated from the gene ORF region is shown in SEQ ID NO.3: SEQ ID NO.3: - The amino acids at positions 33-40 (peptide 1), 221-234 (peptide 2), and 360-372 (peptide 3) correspond to important structures in the HSP70 protein family and play an important role in the stress resistance process. However, experiments have shown that heating the amino acid D (aspartic acid) at position 224 in peptide chain 2 will cause the denaturation of the protein, destroying its secondary and tertiary structures, while Q (glutamine) can form hydrogen bonds through its polar side chain, which helps to maintain the structure of the protein at high temperatures, thereby transforming D into Q. The amino acid P (proline) at position 368 on peptide chain 3 will promote the rotation and structural relaxation of the protein chain at high temperatures, which may lead to the destruction of the secondary and tertiary structures and affect the stability of the protein. The P (proline) at this position is transformed into a more stable C (cysteine), which can enhance the stability of the protein in a hot environment. The function of the protein can be exerted to a greater extent. The modified and optimized amino acid sequence is shown in SEQ ID NO.4, and its corresponding encoding nucleotide sequence is shown in SEQ ID NO.5.

[0019] SEQ ID NO.4: MRCWTALAAIAAVMAVAATAKDKKEEVGTVIGIDLGTTYSCVGVFKNGRVEIIANDQGNRITPSYVAFTADGERLIGDSAKNQLTTNPENTVFDAKRLIGREWTDKSVQHDIQFFPFK VINKNDKPHIKVSTTQGEKVFAAEEXSAMVLGKMKETAEAYLGKTVTHAVVTVPAYFNDAQRQATKDAGTIAGLTVMRIINEPTAAAIAYGIDKKDGEKNILVFQLGGGTFDVSLLTI DSGVFEVVATNGDTHLGGEDFDQRVMDHFIKLYKKKKGKDIRKDNRAVQKLRREVEKAKRSLSSSHQVRIEIESFFEGDDFSETLTRAKFEELNMDLFRSTMKPVQKVLEDSDLQKKEIDEIVLVGGSTRIPCIQQLVKEFFGGKEPSRGINPDEAVAYGAAVQAGVLSGEDDTNDLVL LDVNPLTLGIETVGGVMTKLIPRNTVIPTKKSQIFSTASDNQHTVTIQVFEGERPMTKDNHILGKFDLTGIPPAPRGVPQIEVTFEIDANGILQVSAEDKGTGNKEKITITNDQNRLTPE DIERMIKDAEVFADEDKKLKERVESRNELESYAYSLKNQINDKEKLGAKLSDEDKEKMEEAIEEKIKWLEDNPESDAEDYKTQKKELEDVVQPIITKLYQQSGEAPPPTEDGEDYEKDEL- By transforming aspartic acid into glutamine and proline into cysteine ​​in the amino acid sequence of Bip, its function can be increased to a greater extent on the original basis, thereby improving the thermal stability of the MDH protein.

[0020] 2. Construction and identification of pET-32a-Bip-like recombinant plasmid According to the Lv - Bip-like The full-length gene reading frame sequence ORF was used to design specific primers with restriction endonucleases EcoR I and Xho I as restriction sites, and PrimeSTAR ® The HSDNA Polymerase kit was used to amplify the full length of the Bip-like gene through a PCR instrument. After purification by the gel recovery kit, the product was reacted with the pET-32 a (+) vector at 16 °C overnight under the action of T4 DNA ligase. All ligation products were transformed into Escherichia coli DH5α competent cells, spread on LB plates containing Amp resistance, and placed in a 37 °C constant temperature incubator for 12-16 hours to grow single colonies. The next day, round, plump white monoclonal colonies were picked and placed in Amp-resistant LB culture medium and shaken at 37 °C overnight. The plasmid was extracted using a plasmid mini-extraction kit, and the enzyme digestion products were detected by agarose gel electrophoresis. The successful recombinant plasmid was selected for sequencing. The results of the double enzyme digestion verification experiment of the recombinant vector are shown in the figure. Figure 1 As shown, it proves that the recombinant plasmid was successfully constructed, and the pET-32a-Bip-like recombinant plasmid with correct sequencing results was transformed into the expression strain, and colony PCR was performed for identification.

[0021] 3. Optimized Lv-Bip-like protein induction expression and affinity purification experiments Take out the strain stored in -80 ℃ refrigerator, activate it and inoculate it in 20 ml liquid culture medium with ampicillin, culture it at 37 ℃ and 180 rpm overnight until 14-16 h (OD 600is 0.6-0.8), and the next day, the culture was transferred and 2 ml of bacteria were inoculated into 100 ml of liquid culture medium. After further cultivation for 2 hours, 0.5 mM IPTG was used for induction to express the target protein in large quantities. 1 ml of pre-induction sample was taken for testing. After about 4 hours of induction, the post-induction sample was taken. Then, the cells were centrifuged at 4°C and 8000 rpm for 10 min, the bacteria were collected, and the bacteria were resuspended in sterile PBS. Then, the cells were ultrasonically broken in an ice bath for 30 min, centrifuged, and the supernatant and precipitate were collected for further solubility analysis. The samples were prepared and verified by SDS-PAGE gel electrophoresis. After solubility analysis, the Lv-Bip-like protein appeared in the supernatant and was directly processed by column. The concentration of the purified protein was determined by the Bradford method, and the sample was prepared for electrophoresis verification, and then stored in a -80°C refrigerator for protein function experiment verification. Lane 5 is the purified target protein, and lanes 1-4 are: before induction, after induction, supernatant, and precipitate, respectively. The results of the induction purification of the recombinant protein are shown in the figure. Figure 2 As shown, it is proved that the purified optimized Lv-Bip-like protein is obtained.

[0022] 4. Bradford method to measure Lv-Bip-like protein concentration First, prepare the standard curve: use BSA to prepare BSA solutions of different concentrations, 0.1, 0.08, 0.06, 0.04, 0.02, and 0 mg / ml. Take 50 ml of BSA solutions with different concentration gradients and add them to the enzyme-labeled wells. Then add 200 ml of Bradford Dye Reagent treatment solution, react at room temperature for 10 min, measure the absorbance at 595 nm with the enzyme reader, and record and analyze to prepare the standard curve. Take 50 ml of the purified recombinant protein solution and add it to the enzyme-labeled wells. Then add 200 ml of Bradford Dye Reagent treatment solution, mix with the pipette tip, react at room temperature for 10 min, detect the absorbance, record it, and substitute it into the formula to calculate the recombinant protein concentration. The standard curve and linear equation are shown in Figure 3 As shown, the protein absorbance values ​​are 1.201, 1.119, and 1.304, which are substituted into the linear formula to obtain 1.117 μg / μL.

[0023] 5. In vitro functional experiment of thermal aggregation of Lv-Bip-like protein First, prepare 20 mmol / L Tris-HCl (pH 8.0) buffer and mix malate dehydrogenase (MDH) with recombinant Lv -Bip-like proteins were mixed in a Tris-HCl (20 mmol / L pH 8.0) system at a ratio of 1:2 and heated at 45 °C to remove MDH and Lv-Bip-like protein mixture, reacted for 20 min, and centrifuged at 15000 g, 4 °C for 15 min. Lv -Bip-like protein, containing only MDH. The supernatant and precipitate obtained by centrifugation were prepared separately and analyzed by SDS-PAGE protein electrophoresis.

[0024] Thermal aggregation refers to the changes in the structure and function of proteins or macromolecules caused by increased temperature. It is usually because the high temperature environment destroys the non-covalent interactions of proteins (such as hydrogen bonds, hydrophobic interactions, etc.), causing the proteins to aggregate from the solution into larger complexes.

[0025] The results of MDH thermal aggregation experiment are shown in Figure 4 As shown: In this experiment, the MDH from pig source (pig heart) and the optimized Bip-like protein of Penaeus vannamei were mixed in a Tris-HCl system at a ratio of 1:2, and then the samples in the group without the optimized Bip-like protein were found to be turbid, while the samples in the experimental group with the optimized Bip-like protein were clear and transparent. After SDS-PAGE electrophoresis analysis: After the addition of the optimized Bip-like protein, only a small amount of MDH protein aggregated in the supernatant, while in the control group without the recombinant Bip-like protein, the MDH protein mainly aggregated in the supernatant. This shows that the optimized Lv-Bip-like protein can help improve the thermal stability of MDH, inhibit the thermal aggregation of MDH, and thus protect MDH.

[0026] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. An optimized Lv-Bip-like protein, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

4.

2. The Lv-Bip-like protein according to claim 1, characterized in that The amino acid is obtained by changing the amino acid at position 224 from aspartic acid to glutamine and the amino acid at position 368 from proline to cysteine ​​based on the amino acid sequence shown in SEQ ID NO.

3.

3. The gene encoding the Lv-Bip-like protein according to claim 1, characterized in that: Its nucleotide sequence is shown as SEQ ID NO.

5.

4. Contains Lv - Bip-like A recombinant expression vector of a gene, characterized in that: Said Lv - Bip-like The nucleotide sequence of the gene is shown in SEQ ID NO.

5.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector is a pET-32 a (+) vector.

6. Use of the Lv-Bip-like protein according to claim 1 for improving the heat resistance of malate dehydrogenase.

7. The use according to claim 6, characterized in that: The Lv-Bip-like protein improves the heat resistance of malate dehydrogenase by inhibiting its thermal aggregation.

8. The use according to claim 6, characterized in that: The malate dehydrogenase is derived from pig heart.

9. Use of the Lv-Bip-like protein according to claim 1 in the preparation of an animal feed additive for improving the heat resistance of the protein.

10. The use according to claim 9, characterized in that: The animals are pigs and broiler chickens.

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