Recombinant alpha-1,3-galactosyltransferase and methods of making and using the same

Abstract

The present application relates to the technical field of molecular biology, in particular to a kind of recombinant alpha-1,3-galactosyltransferase and its preparation method and application.The recombinant protein is recombinant alpha-1,3-galactosyltransferase, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO.1.The sequence of alpha-GT gene of the present application realizes the aggregation of high concentration of protein in cell after expression in engineering bacteria, but at the same time, it does not occur with the combination of cell membrane, and this characteristic helps to improve the yield of target protein in the fermentation process of engineering bacteria, reduces the operation difficulty in the process of purification, improves protein purity and activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a recombinant alpha-1,3-galactosyltransferase and a preparation method and application thereof. BACKGROUND

[0002] The immunogenicity of the protein on the surface of the membrane of animal cells is affected by the sequence and spatial structure of the protein itself, and various modifications of the protein glycan are also important reasons for the significant changes in the immunogenicity of the protein. According to previous studies, after the protein is modified by alpha-gal epitopes, the protein is phagocytosed by human immune cells, and the efficiency of antigen presentation and immune response is significantly improved compared with the protein without modification. This technology has been used in clinical application research of using autologous tumor cells to prepare therapeutic vaccines.

[0003] The surface protein of normal human cells does not contain alpha-gal epitope modification groups, and alpha-1,3-galactosyltransferase can be used to modify the protein on the surface of the cell membrane to form alpha-gal epitope modification groups in vitro. By modifying tumor cells in this way and preparing therapeutic vaccines, the body's immune system can be induced to kill residual distant metastatic tumor cells in the body. The modification reaction requires the use of alpha-1,3-galactosyltransferase, so a large amount of alpha-1,3-galactosyltransferase is required, and the use of genetic engineering to construct an alpha-GT high-efficiency expression strain is the simplest and most efficient solution. SUMMARY

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a recombinant alpha-1,3-galactosyltransferase and a preparation method and application thereof.

[0005] The first aspect of the present application provides a recombinant protein, which is a recombinant alpha-1,3-galactosyltransferase, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 1.

[0006] The second aspect of the present application provides a polynucleotide encoding the aforementioned recombinant protein.

[0007] The third aspect of the present application provides an expression vector containing the aforementioned polynucleotide.

[0008] The fourth aspect of the present application provides a host cell containing the aforementioned expression vector or having integrated into the genome an exogenous polynucleotide.

[0009] The fifth aspect of the present application provides a preparation method of the aforementioned recombinant protein, comprising the following steps:

[0010] The aforementioned host cell is cultured under suitable conditions to express the recombinant protein, and the recombinant protein is isolated and purified.

[0011] The sixth aspect of the present application provides use of the aforementioned recombinant protein in preparation of a tumor treatment product.

[0012] The seventh aspect of the present application provides a method for improving the yield of alpha-1,3-galactosyltransferase, comprising the following steps:

[0013] 1) amplifying a genome containing wild alpha-GT protein to obtain a recombinant protein, wherein the nucleotide sequence of the recombinant protein is shown as SEQ ID NO. 1;

[0014] 2) connecting six histidines to the N-terminus of the recombinant protein;

[0015] 3) inserting the product obtained in step 2) into an expression vector framework to obtain an alpha-1,3-galactosyltransferase expression vector;

[0016] 4) introducing the alpha-1,3-galactosyltransferase expression vector into an engineering bacterium for expression.

[0017] The present application has the following beneficial effects: the sequence of the alpha-GT gene of the present application realizes high concentration of aggregation of the protein expressed in the engineering bacterium in the cell, but does not bind to the cell membrane, which helps to improve the yield of the target protein in the fermentation process of the engineering bacterium, reduces the operation difficulty in the purification process, and improves the protein purity and activity. The recombinant protein of the present application is obtained by cutting the alpha-GT gene at a specific site, which can eliminate the transmembrane segment of the target protein, change the original membrane-bound protein into a soluble protein, but does not affect the spatial structure and catalytic activity of the enzyme. The yeast expression system is constructed, the expression element containing the "aldehyde oxidase pressure expression system" is used, the amino acid chain is cut at a specific site when the protein expression is completed, and a soluble expression product is formed in the cell, which can significantly improve the expression amount of the target protein. A specific protein purification process is used to improve the protein purification recovery efficiency.

[0018] The present application determines the gene sequence of the final expressed target protein by comparing the expression efficiency of the target protein of the expression system constructed after cutting the alpha-GT gene sequence at different genetic sites. 1. By comparing the GT protein expression efficiency of the whole gene sequence and the protein transmembrane segment sequence removed by the method, it can be known that the sequence of the present application can effectively improve the expression amount of the protein, but does not affect the spatial structure and biological activity of the enzyme. 2. The expression system constructed with the gene sequence finally accumulates the protein in the form of solubility in the yeast cell (part of the protein is secreted in the form of solubility such as culture supernatant), and the protein does not need to be denatured and renatured in the purification process, which simplifies the protein purification operation and maximally retains the biological activity of the enzyme; 3. Combined with the improvement of the expression system construction and amplification, purification process, the yield of the target protein can be obviously improved from the microgram (ug) level of the product per liter of culture to the milligram (mg) level. The activity of the enzyme is maximally retained, and the activity of the enzyme is consistent with the activity of the SIGMA product of the same type of catalytic glycoarabinose transferase (such as beta-1,4-galactosyltransferase). BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Purification map of ion exchange chromatography Hitrap Q.

[0020] Figure 2 It is the result of SDS-PAGE detection of the protein sample after molecular sieve purification (1 is ion exchange Q2; 2 is ion exchange Q3; 3 is ion exchange Q4; 4 is ion exchange Q5; M is Marker).

[0021] Figure 3 It is the expression vector obtained after adding the alpha-GT gene sequence with 6XHIS sequence to the yeast pPIC-Z-Aa vector.

[0022] Figure 4 Efficiency of CIK cells induced by different methods in killing A549 cells.

[0023] Figure 5 Alpha-gal modified tumor cell vaccine induces specific killing effect. DETAILED DESCRIPTION

[0024] In view of the above considerations in the expression and purification process of alpha-1,3-galactosyltransferase, according to the characteristics of the expression system of Pichia pastoris to foreign proteins, the alpha-GT gene sequence from rat blood cells is optimized, the most suitable gene sequence which ensures the highest expression, purification efficiency and enzyme activity is screened, the special affinity adsorption amino acid sequence gene required for plasmid amplification in E. coli is connected, and then the vector which can express the target protein in Pichia pastoris is further constructed, so as to realize high expression and efficient purification in yeast cells.

[0025] Before particular embodiments of the present application are further described, it is to be understood that the application is not limited to the particular specific embodiments described below; it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting as to the scope of the present application. In this specification and in the claims, unless otherwise indicated, the singular forms "a", "an", and "the" include plural referents.

[0026] When numerical ranges are given, it should be understood that every numerical range encompasses any number falling within the range, unless otherwise indicated. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the present application, unless otherwise stated. Unless otherwise specified, the experimental methods, assays, and preparation methods disclosed herein are carried out using conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.

[0027] Unless otherwise indicated, the experimental methods, assays, and preparation methods disclosed herein are carried out using conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.

[0028] The present application is further described in conjunction with the following specific examples.

[0029] One embodiment of the present application provides a recombinant protein, which is a recombinant alpha-1,3-galactosyltransferase, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 1. Specifically, the recombinant protein is:

[0030] MASMTGGQQMGRDLYDDDDKDRWIHNYQQEEEDTDKEKGREEEQRKEDDTTELRLWDWFNPKKRPEVVTVTKWKAPVVWEGTYNKAILEHYYAKQKITVGLTVFAIGRYIEHYLEEFVTSANRYFMVGHKVIFYVMVDDVSKVPFIELGPLRSFKVFEVKPEKRWQDISMMRMKTIGEHILAHIQHEVDFLFCMDVDQVFQDHFGVETLGQSVAQLQAWWYKADPDDFTYERRKESAAYIPFGQGDFYYHAAIFGGTPIQVLNITQECFKGILLDKKNDIEAEWHDESHLNKYFLLNKPSKILSPEYCWDYHIGLPSDIKTVKLSWQTKEYNLVRKNV

[0031] The nucleotide sequence is shown as SEQ ID NO: 7. Specifically, it is:

[0032]

[0033] Normally, α-GT is widely present in the cell membrane of mammals before apes in evolution, and human cells do not contain α-GT and its gene. In addition, α-GT itself is a membrane protein, and a segment of the amino acid sequence of the enzyme is inserted into the cell membrane in normal cells, which plays a role in fixing the enzyme on the membrane and does not participate in the chemical reaction of enzyme catalysis of galactosyl group transfer. Therefore, part of the active site of the enzyme in the actual catalytic reaction can be expressed and purified by genetic engineering method to improve the efficiency of protein expression and catalytic reaction.

[0034] The present application realizes the aggregation of the protein expressed in the engineered bacteria at a high concentration in the cells by splicing the sequence of the α-GT gene, but at the same time, the combination with the cell membrane does not occur, which helps to improve the yield of the target protein in the fermentation process of the engineered bacteria, reduce the operation difficulty in the purification process, and improve the protein purity and activity.

[0035] Specifically,

[0036] The full gene sequence of α-GT in mammalian cells (wild α-GT sequence) (SEQ ID NO: 3):

[0037] ATGAATGTCAAAGGAAAA GTAATTCTGTCGATGCTGGTTGTCTCAACTGTGATTGTTGTGTTTTGG

[0038] Further, the recombinant protein is further connected with histidine at the N terminal.

[0039] Further, the number of histidine at the N terminal of the recombinant protein is 6.

[0040] Further, the nucleotide sequence of the recombinant protein is shown in SEQ ID NO. 2. Specifically, the nucleotide sequence of the recombinant protein is shown in SEQ ID NO. 2.

[0041] CATCATCATCATCATCAT

[0042] The expressed protein needs a special amino acid sequence as the adsorption site during purification. In this operation, the gene of the expression of 6 consecutive histidine sequences (HIS-HIS-HIS-HIS-HIS-HIS-) is connected to the sequence of the α-GT expression system. The expressed α-GT has 6 consecutive histidines at the N-terminal, forming a special spatial structure, which can be adsorbed by the nickel ions (Ni2+) fixed on the resin. In this way, the target protein is fixed on the resin, and other unnecessary proteins are washed away. After the impurities are washed away, the target protein is separated from the nickel ions by using an eluent, so as to achieve the purpose of purification. The target protein can be adsorbed by the resin containing nickel ions, so as to be separated and purified from the protein crude product of the broken engineering bacteria.

[0043] Optionally, the recombinant protein further comprises an enzyme cutting site, such as an EcoR I enzyme cutting site or an XbaI enzyme cutting site.

[0044] An embodiment of the present application provides a polynucleotide encoding the aforementioned recombinant protein.

[0045] An embodiment of the present application provides an expression vector containing the aforementioned polynucleotide.

[0046] Methods known to those skilled in the art can be used to construct the expression vector. These methods include recombinant DNA technology, DNA synthesis technology, etc. The polynucleotide encoding the recombinant protein can be effectively connected to the multiple cloning site in the vector to guide the synthesis of mRNA and then express the protein, or used for homologous recombination.

[0047] Preferably, the expression vector comprises a start signal element AOX.

[0048] In an embodiment, the expression vector comprises the aforementioned polynucleotide and an expression vector framework selected from the yeast pPIC Z-Aα expression vector framework.

[0049] An embodiment of the present application provides a host cell containing the aforementioned expression vector or the aforementioned polynucleotide integrated into the genome of the host cell.

[0050] The host cell is preferably a yeast cell. The vector used in the method can only be suitable for expression in yeast. Different types of yeast can be used as candidates, but other hosts such as E. coli, plant cells, insect cells, etc. are not suitable for the protein expression and purification process specified in this expression system and method.

[0051] In a preferred embodiment, the host cell is Pichia pastoris X-33 engineering bacteria and / or Pichia pastoris GS-115 engineering bacteria.

[0052] An embodiment of the present application provides a method for preparing the aforementioned recombinant protein, comprising the following steps:

[0053] Culturing the aforementioned host cell under suitable conditions to express the recombinant protein, and then isolating and purifying the recombinant protein.

[0054] An embodiment of the present application provides use of the aforementioned recombinant protein in preparation of a tumor treatment product.

[0055] An embodiment of the present application provides a method for improving yield of α-1,3-galactosyltransferase, comprising the following steps:

[0056] 1) amplifying a genome containing wild α-GT protein to obtain a recombinant protein, wherein the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1;

[0057] 2) connecting 6 histidines to the N-terminus of the recombinant protein;

[0058] 3) inserting the product obtained in step 2) into an expression vector framework to obtain an α-1,3-galactosyltransferase expression vector;

[0059] 4) introducing the α-1,3-galactosyltransferase expression vector into an engineering bacterium for expression.

[0060] The nucleotide sequence of the wild α-GT protein is shown as SEQ ID NO: 3.

[0061] In step 3), the expression vector framework is selected from a yeast pPIC Z-Aα expression vector framework.

[0062] In step 4), the engineering bacterium is selected from Pichia pastoris X-33 engineering bacterium and / or Pichia pastoris GS-115 engineering bacterium.

[0063] Embodiment 1

[0064] 1. Obtain a recombinant α-1,3-galactosyltransferase sequence: amplify from monkey kidney epithelial cells NZP-60 cells, and the amplification primer is

[0065] GT-F: GATCCACAATTATCAACAAGAGGAAGAAGAC (SEQ ID NO: 4)

[0066] GT-R: ATGCAAGCTTCTAGATCAGACATTCTTTCTAACCAAATTATACTCTTTTG (SEQ ID NO: 5).

[0067] The sequence of about 50 nucleotides at the N-terminal of the gene is abandoned, which is the sequence of the part of the protein of the α-GT penetrating the membrane and mainly plays the role of fixing the enzyme protein on the cell membrane in the actual reaction and does not participate in the catalytic activity of the actual galactosyl transfer and connection reaction, so the sequence is abandoned in the construction of the expression system of the α-GT. Under normal circumstances, the α-1, 3-galactosyltransferase with the full-length sequence is normally expressed as a membrane protein, which is a mushroom-like structure, part (i.e. the handle of the mushroom) is inserted into the cell membrane, and the mushroom head part is the part with catalytic activity. To understand which part belongs to the part inserted into the cell membrane, the region of the hydrophilic or hydrophobic characteristics of the amino acid sequence of the expressed protein can be determined. The sequence of the enzyme inserted into the cell membrane is cut off in this method mainly to achieve two purposes, one is to make the expressed enzyme be in a free state instead of being combined with the cell membrane, which can improve the efficiency of separation and purification, and the other is to cut off the sequence without affecting the spatial structure of the part with catalytic activity of the enzyme, but it is beneficial to improve the catalytic efficiency in the reaction.

[0068] 2. Amplification and connection of the desired amino acid sequence in E. coli: The expressed protein needs a special amino acid sequence as the adsorption site for purification. In this operation, the gene of the expression of the sequence of six consecutive histidine sequences (HIS-HIS-HIS-HIS-HIS-HIS-) is connected to the sequence of the α-GT expression system, and the expressed α-GT appears six consecutive histidines at the N-terminal, which can be adsorbed by the resin containing nickel ions, so as to separate and purify the α-GT enzyme protein from the protein crude product of the broken bacteria of the engineering bacteria.

[0069] The sequence of the α-GT (SEQ ID NO: 6) with the sequence of the six HIS sequences (the sequence of the six HIS sequences is from the pRSET-C vector) added at the N-terminal is amplified from the NZP-60 cells and inserted into the E. coli pRSET-C vector.

[0070] (wherein, the bold sequence is EcoR I, the straight line part is 6XHIS , and the curved part is TGA is a termination signal in the sequence of the protein expression gene and does not represent any amino acid. When the ribosome reaches the position of the termination signal during translation, no amino acid can be connected to the amino acid chain before it, the reaction of protein synthesis is automatically terminated, the protein amino acid chain stops elongation, and the reaction is ended.)

[0071] 3. The expression system for high expression in yeast: The sequence with 6X histidine and α-GT gene is amplified in E. coli plasmid, and the part sequence is recovered by purification, enzyme digestion, and then connected into the yeast pPIC Z-Aα expression system. The pPIC Z-Aα plasmid is introduced into Pichia pastoris X-33, GS-115 and other engineering bacteria by electroporation.

[0072] 4. In the pPIC Z-Aα expression system, alcohol oxidase (AOX) and Zeocin resistance gene are co-expressed, so the strain with high Zeocin resistance is also a strain with high AOX expression. 52 strains of each strain are selected to obtain AOX high expression strains on agar plates with different concentrations of Zeocin.

[0073] 5. Large-scale expansion and pressure expression of exogenous protein in yeast engineering bacteria: The selected α-GT high expression strain is expanded in BMMY medium. After 24 hours of culture, 2% volume of methanol is added every 8 hours. Because the expression element of α-GT enzyme and the expression element of alcohol oxidase (AOX) share a promoter, the addition of methanol can induce the increase of AOX expression and also increase the expression of α-GT.

[0074] 6. Cell disruption using high-pressure hydraulic crushing system: After the yeast cell culture expansion reaches the predetermined time, the cell is harvested by centrifugation, and the yeast cell is washed twice with pure water. The cell is resuspended with protein purification adsorption buffer, and the cell suspension is repeatedly pressurized by a high-pressure crusher at 1000-1200 atmospheres. After crushing the cell, high-speed centrifugation is performed to remove the precipitate, and the crude product of the exogenous recombinant protein is obtained.

[0075] 7. The adsorption buffer containing the recombinant protein is passed through a solid-phase chromatography column containing Ni 2+ resin. Most of the recombinant protein containing 6XHIS (HIS-HIS-HIS-HIS-HIS-HIS-) amino acid sequence is adsorbed on the solid phase. The solid-phase column is washed with a washing buffer to remove most of the non-specifically bound other proteins. The eluted protein is passed through a molecular sieve resin column to separate the harvested protein in the eluate according to the molecular weight, and a higher purity recombinant protein is obtained.

[0076] 8. The main function of the recombinant protein obtained by this method is to catalyze the attachment of a galactosyl group to the end of the protein sugar chain to form an α-Gal epitope:

[0077] α1,3GT

[0078] UDP-Gal + Galβ-1,4-GlcNAc-R ————> Galα-1,3-Galβ-1,4-GlucNAc-R + UDP

[0079] The reaction consumes one molecule of UDP-Gal for each molecule of a-Gal epitope product generated. Therefore, by detecting the amount of UDP-Gal consumed per unit time, combined with the amount of enzyme protein, the catalytic activity per unit protein can be calculated. The reaction system containing a-GT and UDP-Gal described above was prepared in 60 tubes, each containing 50 ul, and incubated in a 37C water bath. Every 60 seconds, 2 tubes were taken to a 65C metal bath to heat and inactivate a-GT to terminate the reaction. After all samples were terminated, the concentration of UDP-Gal in each sample was detected by mass spectrometer. The amount of a-Gal epitope catalyzed by unit enzyme protein per unit time was calculated by converting the reduction of UDP-Gal in the sample, and the activity unit was calculated. After the experiment by the above method, the a-GT enzyme activity was calculated to be about 4600 units / mg protein, which is higher than the activity of a similar product β-GT (MERCK SAE0093) (2000 units / mg protein).

[0080] After the above treatment, the results are shown in Figure 1 and Figure 2 The final determination of protein purification reached 4.7 mg of target protein per liter of bacterial solution, and the purity was determined to be 96% by electrophoresis, and the catalytic activity reached 4600 IU / mg. Under the expression system and purification process of the method, the a-GT protein yield reached 4.7 mg of purified protein per liter of bacterial solution, and the actual expression in the experiment could reach more than 10 mg of crude product per liter of culture. The above data is significantly higher than the efficiency of general yeast recombinant protein expression and purification and the catalytic activity of similar commercial glycosyltransferases. It is suggested that the above protein expression and purification process has exact effect on improving the expression, harvesting efficiency and catalytic activity of the protein.

[0081] Example 2 In vitro experimental study on induction of DC-CIK killer cells to kill residual tumor cells in vivo by a-gal modified tumor cell vaccine

[0082] Experimental method:

[0083] 1. Different tumor cells (melanoma A375 cell strain, lung cancer cell strain A549) were reacted with a-GT and UDP-gal to modify the a-gal side chain of SEQ NO: 1 on the antigenic protein on the surface of the tumor cells

[0084] 2. The modified tumor cells and unmodified tumor cells were broken by ultrasonic crushing instrument to prepare cell lysates, and modified melanoma A375 cell vaccine lysate, modified lung cancer cell A549 vaccine lysate, unmodified melanoma A375 cell vaccine lysate and unmodified lung cancer cell A549 vaccine lysate were obtained

[0085] 3, Peripheral venous blood of healthy people is drawn, and mononuclear cells (PBMC) are obtained by density centrifugal separation

[0086] 4, PBMC is incubated in a 6-well culture plate + complete culture solution (RPMD1640 + 10% fetal bovine serum + interleukin-2) for 6 hours, and the suspended part (CIK) and the adherent part (DC) are continuously cultured in different containers

[0087] 5, Modified melanoma A375 cell vaccine lysate, modified lung cancer A549 cell vaccine lysate, unmodified melanoma A375 cell vaccine lysate, and unmodified lung cancer A549 cell vaccine lysate are added to different DC culture holes, and mixed and cultured for 2 days

[0088] 6, The DC stimulated by different vaccine lysates in step 5 is added to CIK, and the culture is continued for 1 week

[0089] 7, Different groups of CIK do killing experiments of tumor cells (using LDH-Cytox Assay Kit produced by BioLegend Company, Biolegend Cat No 426401, and the experimental method is referred to the instruction manual of the kit; the target is counted, and the killing efficiency can be calculated. In unit time, how many percentage of target cells are killed, the killing efficiency of the detected cells can be calculated. The positive control is the result value when all target cells are killed, and the maximum value is 4. The killing efficiency of other cells can be obtained by comparing with the positive cells), and the killing effects of CIK stimulated by modified and unmodified tumor vaccines on the same tumor cells and different tumor cells are compared. The experimental results are as follows:

[0090] 1, As shown in Figure 4 , the laboratory data show that the tumor cell vaccine modified by α-gal can induce CIK with enhanced killing efficiency of tumor cells, and the efficiency is increased by about 40%. That is, the CIK induced by the tumor cell vaccine lysate of α-gal modified A549 cells can kill unmodified A549 cells with an efficiency increased by about 40% compared with the CIK stimulated by the unmodified tumor vaccine.

[0091] 2, As shown in Figure 5As shown, the vaccine prepared from the tumor cell strain modified by α-gal induces DC to stimulate CIK, and the killing efficiency of the same kind of tumor cells is about 30% higher than that of different kinds of tumor cells. That is, the CIK produced by the DC induced by the tumor cell vaccine lysate prepared from the α-gal modified A549 cells kills the unmodified A549 cells with an efficiency about 30% higher than that of the unmodified A375 cells. Similarly, the CIK produced by the DC induced by the tumor cell vaccine lysate prepared from the α-gal modified A375 cells kills the unmodified A375 cells with an efficiency about 30% higher than that of the CIK prepared in the same way and killing the A549 cells.

[0092] Experimental conclusion:

[0093] 1. The vaccine prepared from the same kind of tumor cell strain modified by α-gal has enhanced killing efficiency of the CIK produced by the DC, which indicates that the α-gal modification can enhance the immunogenicity of the tumor-specific antigens on the surface of the tumor cells.

[0094] 2. The tumor cell vaccine prepared from the α-gal modified tumor cells has tumor type specificity in the killing of the tumor cells by the DC-CIK system, that is, the CIK produced by the modified melanoma vaccine has a killing effect on the melanoma cells obviously higher than that on other types of tumor cells. This experimental result is the theoretical basis for using the α-gal modified whole cell vaccine prepared from the autologous tumor tissue as a means for treating the residual tumor in vivo, and is the experimental basis for using the autologous tumor tissue to have a higher efficiency than using other tumor cell strains as the vaccine.

[0095] In summary, the present application effectively overcomes the shortcomings in the prior art and has a high industrial utilization value.

[0096] The above examples only illustratively explain the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application. SEQUENCE LISTING <110> Shanghai Mingda Biotechnology Co., Ltd. <120> Recombinant α-1,3-galactosyltransferase and preparation method and application thereof <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 338 <212> PRT <213> Artificial Sequence <400> 1 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp 1 5 10 15 Asp Asp Asp Lys Asp Arg Trp Ile His Asn Tyr Gln Gln Glu Glu Glu 20 25 30 Asp Thr Asp Lys Glu Lys Gly Arg Glu Glu Glu Gln Arg Lys Glu Asp 35 40 45 Asp Thr Thr Glu Leu Arg Leu Trp Asp Trp Phe Asn Pro Lys Lys Arg 50 55 60 Pro Glu Val Val Thr Val Thr Lys Trp Lys Ala Pro Val Val Trp Glu 65 70 75 80 Gly Thr Tyr Asn Lys Ala Ile Leu Glu His Tyr Tyr Ala Lys Gln Lys 85 90 95 Ile Thr Val Gly Leu Thr Val Phe Ala Ile Gly Arg Tyr Ile Glu His 100 105 110 Tyr Leu Glu Glu Phe Val Thr Ser Ala Asn Arg Tyr Phe Met Val Gly 115 120 125 His Lys Val Ile Phe Tyr Val Met Val Asp Asp Val Ser Lys Val Pro 130 135 140 Phe Ile Glu Leu Gly Pro Leu Arg Ser Phe Lys Val Phe Glu Val Lys 145 150 155 160 Pro Glu Lys Arg Trp Gin Asp lie Ser Met Met Arg Met Lys Thr lie 165 170 175 Gly Glu His lie Leu Ala His lie Gin His Glu Val Asp Phe Leu Phe 180 185 190 Cys Met Asp Val Asp Gin Val Phe Gin Asp His Phe Gly Val Glu Thr 195 200 205 Leu Gly Gin Ser Val Ala Gin Leu Gin Ala Trp Trp Tyr Lys Ala Asp 210 215 220 Pro Asp Asp Phe Thr Tyr Glu Arg Arg Lys Glu Ser Ala Ala Tyr lie 225 230 235 240 Pro Phe Gly Gin Gly Asp Phe Tyr Tyr His Ala Ala lie Phe Gly Gly 245 250 255 Thr Pro lie Gin Val Leu Asn lie Thr Gin Glu Cys Phe Lys Gly lie 260 265 270 Leu Leu Asp Lys Lys Asn Asp lie Glu Ala Glu Trp His Asp Glu Ser 275 280 285 His Leu Asn Lys Tyr Phe Leu Leu Asn Lys Pro Ser Lys lie Leu Ser 290 295 300 Pro Glu Tyr Cys Trp Asp Tyr His Ile Gly Leu Pro Ser Asp Ile Lys 305 310 315 320 Thr Val Lys Leu Ser Trp Gln Thr Lys Glu Tyr Asn Leu Val Arg Lys 325 330 335 Asn Val <210> 2 <211> 1101 <212> DNA <213> Artificial Sequence <400> 2 catcatcatc atcatcatat gaatgtcaaa ggaaaagaat atatcaacag cccagaaggc 60 tctttcttgt ggatatatca ctcaaagaac ccagaagttg atgacagcag tgctcagaag 120 gactggtggt ttcctggctg gtttaacaat gggatccaca attatcaaca agaggaagaa 180 gacacagaca aagaaaaagg aagagaggag gaacaaaaaa aggaagatga cacaacagag 240 cttcggctat gggactggtt taatccaaag aaacgcccag aggttatgac agtgacccaa 300 tggaaggcgc cggttgtgtg ggaaggcact tacaacaaag ccatcctaga aaattattat 360 gccaaacaga aaattaccgt ggggttgacg gtttttgcta ttggaagata tattgagcat 420 gccaaacaga aaattaccgt ggggttgacg gtttttgcta ttggaagata tattgagcat 420tacttggagg agttcgtaac atctgctaat aggtacttca tggtcggcca caaagtcata 480 ttttatgtca tggtggatga tgtctccaag gcgccgttta tagagctggg tcctctgcgt 540 tccttcaaag tgtttgaggt caagccagag aagaggtggc aagacatcag catgatgcgt 600 atgaagacca tcggggagca catcttggcc cacatccaac acgaggttga cttcctcttc 660 tgcatggatg tggaccaggt cttccaagac cattttgggg tagagaccct gggccagtcg 720 gtggctcagc tacaggcctg gtggtacaag gcagatcctg atgactttac ctatgagagg 780 cggaaagagt cggcagcata tattccattt ggccaggggg atttttatta ccatgcagcc 840 atttttggag gaacaccgat tcaggttctc aacatcaccc aggagtgctt taagggaatc 900 ctcctggaca agaaaaatga catagaagcc gagtggcatg atgaaagcca cctaaacaag 960 tatttccttc tcaacaaacc ctctaaaatc ttatctccag aatactgctg ggattatcat 1020 ataggcctgc cttcagatat taaaactgtc aagctatcat ggcaaacaaa agagtataat 1080 ttggttagaa agaatgtctg a 1101 <210> 3 <211> 1131 <212> DNA <213> Artificial Sequence <400> 3 atgaatgtca aaggaaaagt aattctgtcg atgctggttg tctcaactgt gattgttgtg 60 ttttgggaat atatcaacag cccagaaggc tctttcttgt ggatatatca ctcaaagaac 120 ccagaagttg atgacagcag tgctcagaag gactggtggt ttcctggctg gtttaacaat 180 gggatccaca attatcaaca agaggaagaa gacacagaca aagaaaaagg aagagaggag 240 gaacaaaaaa aggaagatga cacaacagag cttcggctat gggactggtt taatccaaag 300 aaacgcccag aggttatgac agtgacccaa tggaaggcgc cggttgtgtg ggaaggcact 360 tacaacaaag ccatcctaga aaattattat gccaaacaga aaattaccgt ggggttgacg 420 gtttttgcta ttggaagata tattgagcat tacttggagg agttcgtaac atctgctaat 480 aggtacttca tggtcggcca caaagtcata ttttatgtca tggtggatga tgtctccaag 540 gcgccgttta tagagctggg tcctctgcgt tccttcaaag tgtttgaggt caagccagag 600 aagaggtggc aagacatcag catgatgcgt atgaagacca tcggggagca catcttggcc 660 cacatccaac acgaggttga cttcctcttc tgcatggatg tggaccaggt cttccaagac 720 cattttgggg tagagaccct gggccagtcg gtggctcagc tacaggcctg gtggtacaag 780 gcagatcctg atgactttac ctatgagagg cggaaagagt cggcagcata tattccattt 840 ggccaggggg atttttatta ccatgcagcc atttttggag gaacaccgat tcaggttctc 900 aacatcaccc aggagtgctt taagggaatc ctcctggaca agaaaaatga catagaagcc 960 gagtggcatg atgaaagcca cctaaacaag tatttccttc tcaacaaacc ctctaaaatc 1020 ttatctccag aatactgctg ggattatcat ataggcctgc cttcagatat taaaactgtc 1080 aagctatcat ggcaaacaaa agagtataat ttggttagaa agaatgtctg a 1131 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <400> 4 gatccacaat tatcaacaag aggaagaaga c 31 <210> 5 <211> 50 <212> DNA <213> Artificial Sequence <400> 5 atgcaagctt ctagatcaga cattctttct aaccaaatta tactcttttg 50 <210> 6 <211> 1057 <212> DNA <213> Artificial Sequence (Artificial Sequence) <400> 6 aattgaattc catcatcatc atcatcatgg tatggctagc atgactggtg gacagcaaat 60 gggtcgggat ctgtacgacg atgacgataa ggatcgatgg atccacaatt atcaacaaga 120 ggaagaagac acagacaaag aaaaaggaag agaggaggaa caaagaaagg aagatgacac 180 aacagagctt cggctatggg actggtttaa tccaaagaaa cgcccagagg ttgtgacagt 240 gaccaaatgg aaggcgccgg ttgtgtggga aggcacttac aacaaagcca tcctagaaca 300 ttattatgcc aaacagaaaa ttaccgtggg gttgacggtt tttgctattg gaagatatat 360 tgagcattac ttggaggagt tcgtaacatc tgctaatagg tacttcatgg ttggccacaa 420 agtcatattt tatgtcatgg tggatgatgt ctccaaggtg ccatttatag agctgggtcc 480 tctgcgttcc ttcaaagtgt ttgaggtcaa gccagagaag aggtggcaag acatcagcat 540 gatgcgtatg aagaccatcg gggagcacat cttggcccac atccaacacg aggttgactt 600 cctcttctgc atggatgtgg accaggtctt ccaagaccat tttggggtgg agaccctggg 660 ccagtcggtg gctcagctac aggcctggtg gtacaaggca gatcctgatg actttactta 720 tgagaggcga aaagagtcgg cagcatatat tccatttggc cagggggatt tttattacca 780 tgcagccatt tttggaggaa cacccattca ggttctcaac atcacccagg agtgctttaa 840 gggaatcctc ctggacaaga aaaatgacat agaagctgag tggcatgatg aaagccacct 900 aaacaagtat ttccttctca acaaaccctc taaaatctla tctccagaat actgctggga 960 ttatcatata ggcctgcctt cagatattaa aactgtcaag ctatcatggc aaacaaaaga 1020 gtataatttg gttagaaaga atgtctgatc tagaaca 1057 <210> 7 <211> 1018 <212> DNA <213> Artificial Sequence <400> 7 atggctagca tgactggtgg acagcaaatg ggtcgggatc tgtacgacga tgacgataag 60 gatcgatgga tccacaatta tcaacaagag gaagaagaca cagacaaaga aaaaggaaga 120 gaggaggaac aaagaaagga agatgacaca acagagcttc ggctatggga ctggtttaat 180 ccaaagaaac gcccagaggt tgtgacagtg accaaatgga aggcgccggt tgtgtgggaa 240 ggcacttaca acaaagccat cctagaacat tattatgcca aacagaaaat taccgtgggg 300 ttgacggttt ttgctattgg aagatatatt gagcattact tggaggagtt cgtaacatct 360 gctaataggt acttcatggt tggccacaaa gtcatatttt atgtcatggt ggatgatgtc 420 tccaaggtgc catttataga gctgggtcct ctgcgttcct tcaaagtgtt tgaggtcaag 480 ccagagaaga ggtggcaaga catcagcatg atgcgtatga agaccatcgg ggagcacatc 540 ttggcccaca tccaacacga ggttgacttc ctcttctgca tggatgtgga ccaggtcttc 600 caagaccatt ttggggtgga gaccctgggc cagtcggtgg ctcagctaca ggcctggtgg 660 tacaaggcag atcctgatga ctttacttat gagaggcgaa aagagtcggc agcatatatt 720 ccatttggcc agggggattt ttattaccat gcagccattt ttggaggaac acccattcag 780 gttctcaaca tcacccagga gtgctttaag ggaatcctcc tggacaagaa aaatgacata 840 gaagctgagt ggcatgatga aagccaccta aacaagtatt tccttctcaa caaaccctct 900 aaaatcttat ctccagaata ctgctgggat tatcatatag gcctgccttc agatattaaa 960 actgtcaagc tatcatggca aacaaaagag tataatttgg ttagaaagaa tgtctgat 1018

Claims

1. A recombinant protein, which is a recombinant α-1, 3-galactosyltransferase, the amino acid sequence of the recombinant protein is shown as SEQ ID NO.

1.

2. The recombinant protein of claim 1, wherein, The recombinant protein is further connected with histidine at its N terminal, and the number of histidine at the N terminal of the recombinant protein is 6.

3. A polynucleotide encoding the recombinant protein of any one of claims 1-2.

4. An expression vector containing the polynucleotide of claim 3.

5. The expression vector of claim 4, wherein, Further comprising one or more of the following features: 1) the expression vector comprises a start signal element AOX; 2) the expression vector comprises the polynucleotide of claim 3 and an expression vector framework, and the expression vector framework is selected from the group consisting of a yeast pPICZ-Aa expression vector framework.

6. A host cell containing the expression vector or the polynucleotide of any one of claims 4-5 integrated into its genome.

7. The host cell of claim 6, wherein The host cell is Pichia pastoris X-33 engineering bacteria and / or Pichia pastoris GS-115 engineering bacteria.

8. The method for preparing the recombinant protein according to any one of claims 1-2, characterized in that, Comprising the following steps: Culturing the host cell of claim 7 under suitable conditions to express the recombinant protein, and then isolating and purifying the recombinant protein.

9. Use of the recombinant protein of any one of claims 1-2 in the preparation of a tumor treatment product, and the tumor treatment product is selected from the group consisting of a tumor cell vaccine modified with α-gal epitopes.

10. A method for increasing the production of α-1, 3-galactosyltransferase, comprising the following steps: 1) amplifying the genome containing wild α-GT protein to obtain a recombinant protein, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1; 2) connecting 6 histidines to the N terminal of the recombinant protein; 3) inserting the product obtained in step 2) into an expression vector framework to obtain an α-1, 3-galactosyltransferase expression vector; 4) introducing the α-1, 3-galactosyltransferase expression vector into engineering bacteria for expression.

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