CN110791513B - Codon-optimized holothuria leucospilota alpha-amylase gene, recombinant expression vector and application thereof - Google Patents
Codon-optimized holothuria leucospilota alpha-amylase gene, recombinant expression vector and application thereof Download PDFInfo
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- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
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Abstract
The invention provides a codon-optimized holothuria leucospilota alpha-amylase gene, a recombinant expression vector and application thereof. On the basis of the gene sequence of the holothuria leucospilota alpha-amylase, the codons of the first 20 amino acids behind the signal peptide of the gene are optimized, and the nucleotide sequence of the optimized holothuria leucospilota alpha-amylase gene is shown as SEQ ID NO. 1. The amino acid sequence coded by the optimized holothuria leucospilota amylase gene sequence is consistent with the code of the original gene sequence, but the protein with alpha-amylase activity can be successfully expressed in a prokaryotic expression system (escherichia coli BL 21). And the non-optimized alpha-amylase sequence cannot be expressed in a prokaryotic expression system.
Description
Technical Field
The invention relates to the technical field of gene modification and protein expression, in particular to a codon-optimized holothuria leucospilota alpha-amylase gene, a recombinant expression vector and application thereof.
Background
Amylase is a hydrolase and is the most widely used enzyme in the fermentation industry at present. Amylases generally act on alpha-1, 4-glucans such as soluble starch, amylose, and glycogen to hydrolyze the alpha-1, 4-glucosidic bonds. Amylases can be classified into alpha-amylases and beta-amylases, depending on the mode of action. The alpha-amylase can hydrolyze alpha-1, 4-glucosidic bonds in the starch and cut the starch into short-chain dextrin with different lengths and a small amount of low-molecular saccharides, so that the viscosity of the starch paste is rapidly reduced, the effects of reducing the consistency and liquefying are achieved, and the capacities of digesting food and absorbing nutrition of animal intestinal tracts are improved. In fish research, alpha-amylase is considered to be a marker for maturation of the digestive capacity of fish larvae. In the early development stage of sea cucumber larvae, amylase can be used as a feed regulator to improve the ingestion, digestion and absorption efficiency of the sea cucumber.
Holothuria leucospilota (Holothuria leucospilota) belongs to the order of tenodactyla (Aspidochirotida), the family of holothuriaceae (Holothuriidae) and the genus Holothuria (Holothuria), and plays an important role in marine ecological restoration. Based on the positive effect of the holothuria leucospilota on promoting the recovery of tropical holothurian resources in China, the research on artificial breeding and cultivation of holothuria leucospilota is developed by the related topic group of the south-sea ocean research institute of Chinese academy of sciences. In order to improve the efficiency of the ingestion, digestion and absorption of the sea cucumber, a subject group tries to obtain the holothuria leucospilota alpha-amylase by a prokaryotic expression method. However, the alpha-amylase gene of holothuria leucospilota cannot be successfully expressed in a prokaryotic expression system, so that the alpha-amylase of holothuria leucospilota needs to be expressed in the prokaryotic expression system by means of a gene modification method.
Among the numerous genetic modification techniques, codon optimization is currently the more widely used technique. Codon optimization is based on the principle that amino acids have codon degeneracy, and ensures that the expressed protein and the endogenously secreted protein have the same physicochemical properties by changing one or more codons in the gene sequence which are suitable for expression in a prokaryotic expression system without changing the original amino acid sequence.
Disclosure of Invention
The invention aims to provide a codon-optimized holothuria leucospilota alpha-amylase gene, a recombinant expression vector and application thereof. The alpha-amylase gene of the holothuria leucospilota successfully expresses protein with alpha-amylase activity in a prokaryotic expression system through codon optimization, thereby providing a feed regulator for artificial breeding of the holothuria leucospilota and improving the ingestion, digestion and absorption efficiency of the holothuria leucospilota.
In order to realize the aim, the invention is based on partial sequence of alpha-amylase gene in the holothuria leucospilota second generation transcriptome database constructed by the subject groupThe full-length sequence of the gene is obtained by RACE technique and then stored in NCBI database (https://www.ncbi.nlm.nih.gov/orffinder/) The Open Reading Frame (ORF) is predicted. The result shows that the amylase gene has a full-length cDNA of 1734bp, an Open Reading Frame (ORF) of 1578bp, 525 amino acids are coded, and the predicted molecular weight is 59.34 KDa. The protein contains a signal peptide and an amylase functional domain at the N-terminus, including a catalytically active site (fig. 1).
Based on the alpha-amylase gene sequence of the holothuria leucospilota shown in figure 1, the codons of the first 20 amino acids behind the signal peptide of the gene are optimized. The method comprises the following specific steps: (a) (ii) replacing the amino acid codons TTC at positions 2 and 17 with TTT; (b) replacing the 6 th amino acid codon GCC with GCG; (c) (ii) replacing the amino acid codon GTT at position 7 with GTG; (d) the 8 th amino acid codon GGA is replaced by GGC; (e) the 10 th amino acid codon CGT is replaced by CGC; (f) (ii) substitution of the amino acid codon at position 12, ACA, to ACC; (g) replacing the 13 th amino acid codon ATA with ATT; (h) replacing the 16 th amino acid codon CTT with CTG; (i) the 18 th amino acid codon AGT is replaced by AGC.
Namely: optimization of the pre-alpha-amylase protein coding gene sequence (first 20 amino acid gene coding sequence after signal peptide): CAG TTC GAT ACC AAC GCC GTT GGA GAT CGT GAA ACA ATA GTG CAG CTT TTC AGT TGG AAA, amino acid sequence: QFDTNAVGDRETIVQLFSWK are provided.
Optimized alpha-amylase protein coding gene sequence (first 20 amino acid gene coding sequences after signal peptide, optimized codons in bold type): CAG TTT GAT ACC AAC GCG GTG GGC GAT CGC GAA ACC ATT GTG CAG CTG TTT AGC TGG AAA, amino acid sequence QFDTNAVGDRETIVQLFSWK.
Specifically, the codon-optimized holothuria leucospilota alpha-amylase gene provided by the invention has a nucleotide sequence shown in SEQ ID No. 1.
A group of primers for amplifying the codon-optimized holothuria leucospilota alpha-amylase gene comprises the following primers:
AMY-CO-F1：5'-TGGAAATGGACAGACGTAGCTCTT-3'；
AMY-CO-F2：5'-CAGTTTGATACCAACGCGGTGGGCGATCGCGAAACCATTGTGCAGCTGTTTAGCTGGAAATGGACAGACGTAGCTCTT-3'；
A-Xho1-R：5'-GTGGTGGTGGTGGTGCTCGAGTCTATATGAAAGAAAGTGAC-3'；
the primers AMY-CO-F1 and A-Xho1-R are used for editing the alpha-amylase gene, and the primers AMY-CO-F2 and A-Xho1-R are used for obtaining the alpha-amylase codon optimization sequence.
A recombinant expression vector contains the codon-optimized holothuria leucospilota alpha-amylase gene.
Preferably, the expression vector of the recombinant expression vector is pET28a plasmid.
A recombinant gene engineering bacterium, the recombinant expression vector.
Preferably, the host bacterium of the recombinant genetic engineering bacterium is escherichia coli BL 21.
The invention also provides a construction method of the recombinant gene engineering bacterium, which comprises the following steps:
cloning the codon-optimized holothuria leucospilota alpha-amylase gene to pET28a plasmid to obtain a recombinant expression vector, and transforming the recombinant expression vector into escherichia coli BL21 to obtain the recombinant genetic engineering bacteria.
Compared with the prior art, the invention has the advantages that:
compared with amylase from other animals, plants or bacteria, the amylase expressed by the holothuria gene is more beneficial to improving the feeding and digestion absorption efficiency of artificially cultured holothuria by being used as a feed regulator, the codon optimization technology is utilized to modify the alpha-amylase gene of holothuria leucospilota, the amino acid sequence coded by the optimized holothuria leucospilota amylase gene sequence is consistent with the code of the original gene sequence, but the protein with the alpha-amylase activity can be successfully expressed in a prokaryotic expression system (escherichia coli BL 21). Whereas the non-optimized alpha-amylase sequence could not be expressed in a prokaryotic expression system (fig. 4). The invention is expected to provide a feed regulator which is beneficial to ingestion and digestion for artificially cultured sea cucumbers.
Drawings
FIG. 1 shows the nucleotide and amino acid sequences and codon optimization site information of the holothuria leucospilota alpha-amylase gene.
FIG. 2 shows the gene cutting sequence of the alpha-amylase of holothuria leucospilota obtained by PCR amplification using primers AMY-CO-F1 and A-Xho 1-R;
FIG. 3 is an optimized sequence of the alpha-amylase gene of holothuria leucospilota obtained by PCR amplification using primers AMY-CO-F2 and A-Xho1-R, wherein the bold font is a sequence containing optimized codons, and the gray scale brightness is optimized codons;
FIG. 4 is a graph showing that the non-optimized alpha-amylase sequence cannot be expressed in a prokaryotic expression system. The left side shows the expression condition of the holothuria leucospilota alpha-amylase recombinant protein with the unoptimized codon for 0,1,2,4 and 8 hours after the addition of 1.0mM IPTG, and the right side shows the expression condition of the holothuria leucospilota alpha-amylase recombinant protein with the unoptimized codon for 4 hours after the addition of IPTG to the final concentration of 0.1,0.3,0.5,0.7 and 1.0mM, and no target band with the size of 59kDa appears.
FIG. 5 shows prokaryotic expression and purification of codon-optimized sequences of holothuria leucospilota alpha-amylase gene. A is the expression condition of alpha-amylase recombinant protein of the holothuria leucospilota induced by adding IPTG for 0,1,2,4 and 8 hours, B is the expression condition of alpha-amylase recombinant protein of the holothuria leucospilota added with IPTG to the final concentration of 0.1,0.3,0.5,0.7 and 1.0mM, C is the purification of alpha-amylase recombinant protein, and M represents marker; 1: supernatant induced at 37 ℃; 2: precipitation induced at 37 ℃; 3: 20mM Imidazole eluate fraction; 4-5: 50mM Imidazole eluate fraction; 6: 500mM Imidazole eluted fraction.
FIG. 6 shows the determination of alpha-amylase activity after prokaryotic expression of codon-optimized sequence of holothuria leucospilota alpha-amylase gene. A is a glucose standard curve, B is the activity measurement result of alpha-amylase at different temperatures, and C is the activity measurement result of alpha-amylase at different pH values.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The experimental procedures in the following examples were carried out in a conventional manner or according to the kit instructions unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Primer synthesis and sequencing were performed by Shanghai bioengineering, Inc.
Example 1
1. Obtaining of holothuria leucospilota amylase gene full length and prediction of open reading frame
Partial sequence of alpha-amylase gene is screened from the second generation transcriptome database of holothuria leucospilota constructed by the subject group, and the full-length sequence of the gene is obtained by RACE technology. Then in NCBI database (https:// www.ncbi.nlm.nih.gov/orffinder/) The Open Reading Frame (ORF) is predicted. The result shows that the amylase gene has a full-length cDNA of 1734bp, an Open Reading Frame (ORF) of 1578bp, 525 amino acids are coded, and the predicted molecular weight is 59.34 KDa. The protein contains a signal peptide and an amylase functional domain at the N-terminus, including a catalytically active site (fig. 1).
2. Optimization of holothuria leucospilota alpha-amylase gene codon and construction of protein expression vector
2.1 optimization of the codon of the Alpha-amylase Gene of Holothuria leucospilota
2.1.1 editing quasi-optimized sequence of holothuria leucospilota alpha-amylase
(1) Extraction of intestine tissue DNA of Jatropha curcas
Taking intestine tissue of Jatropha curcas, extracting the intestine tissue genome DNA of Jatropha curcas by adopting a marine animal tissue genome DNA extraction kit (Tiangen Biochemical technology Co., Ltd., Beijing), and strictly performing the operation steps according to the instruction. Genomic DNA quantification was performed using NanoDropTM2000 spectrophotometer, and the quality is detected by agarose electrophoresis.
(2) Design of sequence editing primers
Based on the obtained ORF of the α -amylase gene, primers AMY-CO-F1 and A-Xho1-R (Table 1) were designed using Primer Premier 5 software for the purpose of clipping the gene sequence corresponding to 20 amino acids after the ORF signal peptide was cut off. For the subsequent construction of prokaryotic expression vectors, A-Xho1-R contains a sequence capable of homologous recombination with pET28a vector and contains a cleavage site Xho-I.
(3) PCR amplification
Taking the genomic DNA of intestinal tissues of the Hojothuria leucospilota as a template, and carrying out PCR amplification by using primers AMY-CO-F1 and A-Xho1-R, wherein the reaction program is as follows: pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 sec, annealing at 56 ℃ for 30 sec, extension at 72 ℃ for 1.5 min, 35 cycles.
The clipped holothuria leucospilota alpha-amylase gene sequence (shown as SEQ ID NO. 2) is obtained by the PCR amplification.
2.1.2 obtaining optimized sequence of alpha-amylase gene from Holothuria leucospilota
(1) Primer design
Primer AMY-CO-F2 (Table 1) was designed, containing optimized codons and a sequence partially identical to AMY-CO-F1.
(2) PCR amplification
PCR amplification is carried out by using primers AMY-CO-F2 and A-Xho1-R and taking a PCR product (clipped holothuria leucospilota alpha-amylase gene sequence diluted by 100 times) obtained by 2.1.1(3) PCR amplification as a template, and the reaction program is as follows: pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 sec, annealing at 60 ℃ for 30 sec, extension at 72 ℃ for 2 min, 35 cycles.
Through the PCR amplification, the optimized sequence of the alpha-amylase gene codon of the holothuria leucospilota (shown as SEQ ID NO. 1) is obtained. The sequence is changed with the alpha-amylase gene sequence of the holothurian shown in figure 1 as follows: (a) (ii) replacing the amino acid codons TTC at positions 2 and 17 with TTT; (b) replacing the 6 th amino acid codon GCC with GCG; (c) (ii) replacing the amino acid codon GTT at position 7 with GTG; (d) the 8 th amino acid codon GGA is replaced by GGC; (e) the 10 th amino acid codon CGT is replaced by CGC; (f) (ii) substitution of the amino acid codon at position 12, ACA, to ACC; (g) replacing the 13 th amino acid codon ATA with ATT; (h) replacing the 16 th amino acid codon CTT with CTG; (i) the codon AGT for the amino acid at position 18 is replaced by AGC as shown in FIGS. 2 and 3.
2.2 construction of holothuria leucospilota alpha-amylase protein expression vector
(1) Primer design
(2) PCR amplification
PCR amplification is carried out by taking a PCR product (optimized codon sequence of holothuria leucospilota alpha-amylase gene diluted by 100 times) obtained by 2.1.2(2) PCR amplification as a template and utilizing primers AMY-CO-F3 and A-Xho1-R, wherein the reaction program is as follows: pre-denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 sec, annealing at 60 ℃ for 30 sec, extension at 72 ℃ for 2 min, and 35 cycles to obtain PCR products.
(3) Prokaryotic expression vector construction
And (2) recovering and purifying a PCR product obtained by the PCR amplification of 2.2(2), and then constructing a pET28a vector by utilizing a homologous recombination method, specifically, preparing a linearized pET28a vector with Xho-I and Sac-I enzyme cutting sites, and then carrying out homologous recombination with a holothuria leucospilota amylase target gene containing two identical enzyme cutting sites and an optimized codon according to a certain ratio under a water bath condition of 16 ℃ to obtain a holothuria leucospilota alpha-amylase recombinant expression plasmid with the optimized codon [ namely, inserting the codon optimized sequence (the nucleotide sequence is shown as SEQ ID NO. 1) of the holothuria leucospilota alpha-amylase gene between the Xho-I and Sac-I enzyme cutting sites of the pET28a vector ], and then transforming the recombinant expression plasmid into a BL21 escherichia coli expression system.
TABLE 1 primer table for codon optimization and prokaryotic expression vector construction of holothuria leucospilota alpha-amylase
3. Expression purification and activity detection of holothuria leucospilota alpha-amylase protein
3.1 expression and purification of holothuria leucospilota alpha-amylase protein
After sequencing verification, the constructed codon-optimized holothuria leucospilota alpha-amylase recombinant expression plasmid is transferred into host escherichia coli BL21(DE 3); randomly picking 5 monoclonal colonies, respectively inoculating to 5mL of 1 μ g/mLKan + LB liquid medium, culturing at 37 deg.C and 250rpm overnight (12-16 hr); inoculating the overnight cultured bacterial liquid into a fresh Kan + LB liquid culture medium according to the ratio of 1:50v/v, and culturing at 37 ℃ and 250rpm until the OD600 is about 0.6; adding IPTG to a final concentration of 1.0mM to induce 0,1,2,4,8 hours respectively at 30 ℃ and 200 rpm; or adding IPTG to final concentration of 0.1,0.3,0.5,0.7,1.0mM, 30 deg.C, 200rpm, and inducing for 4 hr; to determine the optimal IPTG concentration and induction time, 1000Xg, centrifuging for 1min, collecting thalli and storing at 4 ℃; adding 50 mu L of double distilled water into the collected thalli, uniformly mixing by vortex, taking 10 mu L of the double distilled water, adding an equal volume of 2 XLoadingbuffer, and boiling in boiling water for 3-5 minutes to obtain a protein sample; and (3) carrying out SDS-PAGE protein electrophoresis analysis on the treated protein sample, and detecting the expression condition of the alpha-amylase recombinant protein of the holothuria leucospilota, wherein the result is shown in figure 5. The results of using the non-optimized alpha-amylase as a control (i.e., replacing the codon optimized sequence of the holothuria leucospilota alpha-amylase gene with the coding sequence of the non-optimized alpha-amylase to construct a prokaryotic expression vector which is the same as the codon optimized sequence of the holothuria leucospilota alpha-amylase gene, and performing the expression and purification steps of alpha-amylase protein) show that the non-optimized alpha-amylase sequence cannot be expressed in a prokaryotic expression system (fig. 4).
The IPTG induction concentration is determined to be 1mM, the induction time is determined to be 4 hours, and the bacterial liquid obtained under the induction condition is subjected to the following experiments:
collecting crude protein: the cells were dissolved in a buffer (8M Urea,50mM Tris-HCl,300mM NaCl, 0.1% Triton X-100, pH 8.0), sonicated, and centrifuged to collect crude supernatant proteins. Balancing: taking 5mLNi-NTA, washing the equilibrium column by using a Binding buffer with 5 times of the volume of the column bed, and enabling the flow rate to be 5 mL/min; and (3) incubation: incubating the crude protein with the balanced column packing for 1 h; column mounting: putting the incubated product on a column, and collecting and flowing out; balancing: washing the equilibrium column with a Binding buffer; impurity washing: washing the column with Washingbuffer and collecting the effluent; and (3) elution: eluting with Elutionbuffer, and collecting the effluent; and (3) purification and detection: and respectively processing the crude protein, the eluate of the washing impurities and the eluate of the elution impurities, preparing samples and preparing SDS-PAGE for detection. And (3) dialysis concentration: dialyzing the eluted and collected components into a solution containing 50mM Tris, 300mM NaCl and pH8.0, concentrating by using PEG20000 after dialysis, filtering by using a 0.22 mu m filter membrane, and subpackaging by 1mL/tube for storage at-80 ℃ to obtain the purified holothuria leucospilota alpha-amylase.
3.2 Activity detection of Jazu holothurian alpha-amylase protein
The activity of the purified holothuria leucospilota alpha-amylase is detected by utilizing an alpha-amylase (alpha-AL) activity detection kit (purchased from Beijing Soilebao science and technology Co., Ltd., model BC0615), the specific detection method refers to the specification of the kit, then a glucose standard curve is made, and the activity of the amylase under different pH and different temperature conditions is detected. One unit of enzyme activity is defined as: the amount of enzyme required to produce 1mol of glucose per minute at the optimum temperature (50 ℃) and pH (6.0) is defined as one unit of enzyme activity. The absorbance of the holothuria leucospilota alpha-amylase at 562nm (A562) is A562-0.836, and the standard curve y is substituted into 1.966x +0.2246 (R)20.997), and the calculated activity of the holothuria leucospilota alpha-amylase is 62.2U/mg, and the result is shown in figure 6.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> codon-optimized holothuria leucospilota alpha-amylase gene, recombinant expression vector and application thereof
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ttgatagttg attgtatacg taggcacact cccttgaaag atttcgggag gtttaatttc 780
gccgagtctt gggagctcct tcccagtggc gaagctgtaa gttttattga caaccatgat 840
aatcagagag gagaaggcca agaagagatt gtgaatttta aagaacccaa ggaatacaaa 900
atggctaacg ctctcatgct ggcgtggcct tacggtatca acagggtcat gtcaagttat 960
gaatttgaga catctgacga tggacctccg tctaatgaag acggcgatct tctgtcgccc 1020
gaaatagatg aagacggctt atgtaccgga ggttgggtat gcgaacaccg atggagggtg 1080
atcaaaaata tggtcaagta ccagaacgtg gtgcgcaaag aatctgttat gaattggtgg 1140
gacaatggaa atcaacaagt ggcattcggt cgaggcaaaa aggggttttt tgtaatgaat 1200
aatgaactcg aacagaatct gaccgagacc ataatgacag gcctaccaca gggtgaatac 1260
tgtaatgtaa tattaggtga aatgaccgat ggcgagtgtt cgggaccaac tgtacaagtc 1320
aactctgaag gttacgccga cttcaccatt gcttttgatt ccgaagaacc tatggttgcc 1380
atccatgttg atgccctggt cgctggtacg ggaaacgtac atgttgcttc ctttgtggta 1440
atactgttgg cgttcctcct gtcaccttct ttcatatag 1479
Claims (8)
1. A codon-optimized holothuria leucospilota alpha-amylase gene is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. A group of amplification primers of the codon-optimized Holothuria leucospilota alpha-amylase gene, which are disclosed by claim 1, and are characterized by comprising the following primers:
AMY-CO-F1：5'-TGGAAATGGACAGACGTAGCTCTT-3'；
AMY-CO-F2：5'-CAGTTTGATACCAACGCGGTGGGCGATCGCGAAACCATTGTGCAGCTGTTTAGCTGGAAATGGACAGACGTAGCTCTT-3'；
A-Xho1-R：5'-GTGGTGGTGGTGGTGCTCGAGTCTATATGAAAGAAAGTGAC-3'；
the primers AMY-CO-F1 and A-Xho1-R are used for editing the alpha-amylase gene, and the primers AMY-CO-F2 and A-Xho1-R are used for obtaining the alpha-amylase codon optimization sequence.
3. A recombinant expression vector comprising the codon-optimized Holothuria leucospilota alpha-amylase gene of claim 1; the expression vector of the recombinant expression vector is pET28a plasmid.
4. A recombinant genetically engineered bacterium comprising the recombinant expression vector of claim 3.
5. The recombinant genetically engineered bacterium of claim 4, wherein the host bacterium of the recombinant genetically engineered bacterium is Escherichia coli BL 21.
6. The construction method of the recombinant genetically engineered bacterium of claim 5, comprising the steps of:
cloning the codon-optimized holothuria leucospilota alpha-amylase gene of claim 1 to pET28a plasmid to obtain a recombinant expression vector, and transforming the recombinant expression vector into escherichia coli BL21 to obtain recombinant genetic engineering bacteria.
7. Use of the codon-optimized holothuria leucospilota alpha-amylase gene according to claim 1 or the recombinant expression vector according to claim 3 in the preparation of alpha-amylase in a prokaryotic expression system.
8. The use according to claim 7, wherein the prokaryotic expression system is E.coli BL 21.
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