CN110923252A

CN110923252A - Codon-optimized housefly acetylcholinesterase gene, protein and application

Info

Publication number: CN110923252A
Application number: CN201911141293.4A
Authority: CN
Inventors: 杜欣军; 贾起起; 李萍; 杜婷; 王硕
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2019-11-20
Filing date: 2019-11-20
Publication date: 2020-03-27

Abstract

The invention relates to a housefly acetylcholinesterase gene optimized by codons, wherein the nucleotide sequence of the housefly acetylcholinesterase gene is SEQ ID No. 3. The optimized acetylcholinesterase gene has obvious sensitivity to organophosphorus and aminocaproic acid ester pesticides with low concentration, can be used for pesticide residue detection of fruits and vegetables, is simple to operate and low in production cost, and provides a stable and sensitive protease material for developing pesticide residue detection products. The sequence is favorable for expressing housefly acetylcholinesterase in large batch through genetic engineering, and the coded amino acid sequence is unchanged, so that the enzymatic property of the sequence is more stable and is close to that of biologically extracted acetylcholinesterase, convenience is provided for reducing cost and wide application, and the sequence has great application value.

Description

Codon-optimized housefly acetylcholinesterase gene, protein and application

Technical Field

The invention belongs to the technical field of genetic biological engineering, and particularly relates to a housefly acetylcholinesterase gene and protein optimized by codons and application thereof.

Background

The acetylcholinesterase is a key enzyme for biological nerve conduction, can degrade acetylcholine, terminate the excitation of neurotransmitter on postsynaptic membranes, and ensure the normal transmission of nerve signals in organisms.

Research shows that various compounds can inhibit the activity of acetylcholinesterase, and organophosphorus and carbamate pesticides have obvious inhibition effect on the acetylcholinesterase. The organophosphorus pesticide is a pesticide which is widely used in China and has the largest dosage, and the overproof pesticide residue in fruits and vegetables is easy to cause acute organophosphorus pesticide poisoning, further cause cardiac damage and even cause sudden cardiac death. As a big country for producing and consuming fruits and vegetables in China, the problem of pesticide residue is not ignored. Therefore, the use amount of the pesticide is strictly controlled, and the work is well done in the prevention and detection stages to monitor the safety of edible agricultural products.

At present, the pesticide residue detection mainly comprises a chromatographic detection method and a rapid detection method. The chromatographic detection method has high reliability of the detection result, but requires better instruments and equipment, is expensive, has complicated detection steps, and requires professional level of detection personnel, so that the method is inconvenient for carrying out large-scale rough detection on samples. The rapid detection method mainly comprises a living body bioassay method, a biological enzyme method, an immunoassay method and a quick detection method, wherein a colorimetric method in the biological enzyme method detection has high sensitivity, simple and convenient operation and rapid detection, can detect various residual pesticides, but acetylcholinesterase used for detection is easy to inactivate and is difficult to produce and purify. The purity of the acetylcholine esterase from the housefly in the market is low, the enzyme activity of the acetylcholine esterase from the electric eel is high but the price is high, and how to prepare the acetylcholine esterase with stable property and reduce the production cost is a problem to be solved at present. Recombinant expression technology provides an effective way for solving the problem. However, at present, active recombinant enzymes cannot be obtained by using prokaryotic system expression, and the expression efficiency is low by using eukaryotic system expression.

Through searching, the following two patent publications related to the patent application of the invention are found:

1. a mutated housefly acetylcholinesterase gene and its expression (CN101712963A) are disclosed, which is obtained by amino acid site-directed mutagenesis at the 180 th, 327 th and 374 th sites of the amino acid sequence SEQ ID No.1 of the existing housefly acetylcholinesterase gene. The amino acid sequence of the protein coded by the gene is shown as SEQ ID NO.3, the nucleotide sequence is shown as SEQ ID NO.4, and the gene can be quickly and efficiently expressed in a yeast expression system. The mutated acetylcholinesterase gene has obvious sensitivity to organophosphorus and carbamate pesticides with low concentration, can be used for detecting pesticide residues in agricultural products, provides a more sensitive protease material for developing pesticide biological detection products, and has great application value.

2. The mutant housefly acetylcholinesterase gene and the expression vector (CN101709304A) thereof, and the protein expressed by the mutant housefly acetylcholinesterase has amino acid substitutions at the 262 th site, the 327 th site and the 374 th site compared with the prior SEQ ID NO.1 amino acid sequence. The mutated acetylcholinesterase obtained by the invention has obvious sensitivity to organophosphorus and carbamate pesticides with low concentration, can be used for detecting pesticide residues in agricultural products, provides a more sensitive protease material for developing pesticide biological detection products, and has great application value.

By contrast, the present patent application is substantially different from the above patent publications.

Disclosure of Invention

The invention aims to overcome the defects of the environmental stress problem in the prior art and provide a housefly acetylcholinesterase gene and protein which are optimized by codons and application thereof.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a housefly acetylcholinesterase gene optimized by codons has a nucleotide sequence of SEQ ID No. 3.

Moreover, the housefly acetylcholinesterase gene is subjected to codon optimization on the basis of the existing housefly acetylcholinesterase gene, and can encode protein with acetylcholinesterase activity.

Furthermore, the housefly acetylcholinesterase gene is obtained by optimizing 29 codons on the basis of the original gene sequence SEQ ID NO.1, namely ccg → cca cgc, cgg → cga gcg → gct tgc → tgt ata → atc.

The amino acid coded by the codon-optimized housefly acetylcholinesterase gene has a sequence of SEQ ID NO. 2.

An expression vector comprising a housefly acetylcholinesterase gene as described above.

Eukaryotic expression cells comprising an expression vector as described above.

The preparation method of the protein expressed by the housefly acetylcholinesterase gene which is optimized by the codon.

Moreover, the steps are as follows:

⑴ obtaining optimized gene sequence by gene synthesis;

⑵ connecting the segment with acetylcholinesterase gene to plasmid pPIC9K-His to construct the target gene expression vector;

⑶ the plasmid successfully constructed is cut by SalI, and is transferred into pichia pastoris GS115 competent cells by electric shock to obtain an expression strain;

⑷ the correct yeast strain will be verified for induction expression of the protein of interest;

⑸ screening a strain with high expression level to perform induction expression of induction time, inducer concentration and initial pH of culture medium;

⑹ the target protein is induced to express and separate according to the above optimized condition.

The protein expressed by the housefly acetylcholinesterase gene optimized by the codon is applied to pesticide detection.

The use of the protein expressed by the codon-optimized housefly acetylcholinesterase gene as described above.

The invention has the advantages and positive effects that:

1. the recombinant housefly acetylcholinesterase has high sensitivity and stability, can be used as an enzyme source required by a rapid detection method, and the encoded protein has stable enzymology properties.

2. The sequence of the housefly acetylcholinesterase gene is derived from housefly, and the nucleotide sequence of the housefly acetylcholinesterase gene comprises 1737 basic groups and codes 579 amino acids. The nucleotide sequence of the housefly is the same as that of SEQ ID NO.1, and the amino acid sequence is the same as that of SEQ ID NO. 2; the sequence of SEQ ID NO.1 is subjected to codon optimization, the optimized nucleotide sequence is the same as that of SEQ ID NO.3, namely the sequence of the gene of the invention, and the coding and amino acid sequence of the gene are the same as those of SEQ ID NO. 2. By optimizing partial codons, the sequence can be quickly and efficiently expressed in a pichia pastoris expression system, and the expression quantity is improved by 1.3 times. The optimized acetylcholinesterase gene has obvious sensitivity to organophosphorus and aminocaproic acid ester pesticides with low concentration, can be used for pesticide residue detection of fruits and vegetables, is simple to operate and low in production cost, and provides a stable and sensitive protease material for developing pesticide residue detection products. The sequence is favorable for expressing housefly acetylcholinesterase in large batch through genetic engineering, and the coded amino acid sequence is unchanged, so that the enzymatic property of the sequence is more stable and is close to that of biologically extracted acetylcholinesterase, convenience is provided for reducing cost and wide application, and the sequence has great application value.

3. Another technical problem to be solved by the invention is to optimize protein induction expression conditions, including induction time, inducer concentration and initial culture medium concentration, so as to improve the expression amount of acetylcholinesterase in a eukaryotic system, provide reliable technical support for industrial production, and save cost to the maximum extent.

Drawings

FIG. 1 is a diagram showing the results of the verification of 4 recombinant plasmids constructed in the present invention, wherein (a) is a diagram showing the results of colony PCR, (b) is a diagram showing the results of plasmid PCR, and (c) is a diagram showing the results of double digestion;

FIG. 2 is a diagram showing the results of verifying the genomic PCR of the yeast transformants of the present invention, wherein (a) is a diagram of 9 recombinant plasmid yeast transformants and (b) is a diagram of 2 empty plasmid yeast transformants;

FIG. 3 is a diagram showing the screening results of transformants of the high-expression yeast of the present invention, wherein M: marker, 1-9: experimental strain, 10: empty plasmid transformants;

FIG. 4 is a graph showing the trend of protein expression levels when the concentration of the inducer is optimized in the present invention;

FIG. 5 is a graph showing the trend of protein expression levels at the initial pH of the optimal induction medium in the present invention;

FIG. 6 is a graph showing the purification results of a target protein of the present invention, wherein M: marker, 1: crude protein, 2: and (5) after purification.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

Preferably, the housefly acetylcholinesterase gene is subjected to codon optimization on the basis of the existing housefly acetylcholinesterase gene, and can encode protein with acetylcholinesterase activity.

Preferably, the housefly acetylcholinesterase gene is obtained by optimizing 29 codons on the basis of the original gene sequence SEQ ID NO.1, namely ccg → cca cgc, cgg → cga gcg → gct tgc → tgt ata → atc.

Eukaryotic expression cells comprising an expression vector as described above.

The protein expressed by the housefly acetylcholinesterase gene optimized by the codon is applied to the detection of organophosphorus and carbamate pesticides.

The protein expressed by the acetylcholinesterase gene has 579 amino acids, has the amino acid sequence of SEQ ID NO.2 and the nucleotide sequence corresponding to SEQ ID NO.1, namely the original gene sequence of the housefly acetylcholinesterase.

On the basis of the nucleotide sequence of SEQ ID NO.1, the codon optimization is carried out on the nucleotide sequence of SEQ ID NO.1 by analyzing the preference codon of yeast to obtain the housefly acetylcholinesterase optimized gene suitable for yeast expression, which corresponds to the nucleotide sequence of SEQ ID NO. 3. The optimization of 29 codons is carried out, and the following steps are respectively carried out: ccg → cca cgc, cgg → cga gcg → gcttgc → tgt ata → atc.

The preparation method of the housefly acetylcholinesterase gene expression protein with the optimized codon comprises the following steps:

1) obtaining an optimized gene sequence through gene synthesis;

2) connecting the segment with acetylcholinesterase gene to plasmid pPIC9K-His to construct the target gene expression vector;

3) carrying out enzyme digestion on the successfully constructed plasmid by Sal I, and transferring the plasmid into a pichia pastoris GS115 competent cell by electric shock to obtain an expression strain;

4) carrying out induction expression on the yeast strains which are verified to be correct on the target protein;

5) screening a strain with higher expression quantity to perform induction expression of induction time, inducer concentration and initial pH of a culture medium;

6) and performing induced expression and separation of the target protein according to the optimized conditions.

Specifically, the preparation is as follows:

first, obtaining the gene sequence of housefly acetylcholinesterase

Based on the housefly acetylcholinesterase gene sequence, 29 rare codons are optimized, ccg → ccacgc, cgg → cga gcg → gct tgc → tgt ata → atc,

design pPIC9K-His plasmid primers: s2: 5'-GACTGGTTCCAATTGACAAGC-3'

A2：5’-GCAAATGGCATTCTGACATCC-3’

EcoRI and Not I enzyme cutting sites are respectively added before and after the optimized gene sequence, and the optimized gene sequence and the designed primer sequence are sent to a gene company for synthesis.

Second, construction of housefly acetylcholinesterase gene recombinant plasmid AchE-pPIC9K-His

Extracting a plasmid containing housefly acetylcholinesterase genes and a pPIC9K-His plasmid, carrying out double digestion on the plasmid by using EcoR I and Not I, recovering a target fragment, connecting the target fragment with T4DNAligase, carrying out heat shock transformation on the plasmid to an escherichia coli DH5 α competent cell, coating the competent cell on an LB-kana plate at 37 ℃ for overnight culture, selecting positive clones for PCR verification, double digestion verification and sequencing, wherein the PCR result is shown in figure 1, and the 1% agarose gel electrophoresis result shows that bands with expected sizes all appear, and the sequencing result also proves that the recombinant plasmid is successfully constructed.

Thirdly, construction of recombinant expression strains

Carrying out single enzyme digestion on the recombinant plasmid by SalI, uniformly mixing 80 mu L of competent cells and 20 mu L of linearized plasmid, transferring the mixture into a 0.2cm precooled electric shock cup, adding 1mL precooled sorbitol after electric shock transformation, transferring the mixture into a 1.5mL centrifuge tube after uniform mixing, coating each 250 mu L of mixture on an MD-G418 flat plate, and carrying out inverted culture at 28 ℃ for 3-5 days.

Single colonies on the plate were picked, inoculated into 5mLYPD liquid medium, cultured overnight at 28 ℃ and 200rpm, yeast genome was extracted, verified by PCR using plasmid primer S2/A2, and PCR results for yeast genome were shown in FIG. 2, and 1% agarose gel electrophoresis showed that bands of the expected size were present.

Fourth, the induced expression of housefly acetylcholinesterase gene

Selecting 10 positive yeast transformants, inoculating the transformants in 5mLBMGY culture medium, and culturing at 28 ℃ and 220rpm overnight; the overnight suspension was inoculated into 50mL of BMGY medium at 1% inoculum size, shaken at 220rpm at 28 ℃ until OD600 became 2-6, centrifuged to collect the cells, transferred to 100mLBMMY medium, subjected to shaking culture at 220rpm at 28 ℃ for 6 days, added with 1% methanol every 24 hours, and separated 1mL of the medium, precipitated with an equal volume of acetone to precipitate the protein, and subjected to SDS-PAGE. Electrophoresis showed that the recombinant yeast showed a specific band around 68kDa, which is consistent with the size of the target protein (see FIG. 3).

Fifthly, optimizing expression conditions

And selecting a strain with higher expression quantity, and optimizing expression conditions to improve the protein expression quantity.

(1) The inducer concentration is first optimized. Respectively setting the concentrations of the inducers to be 0.5%, 1%, 1.5% and 2%, carrying out inducible expression according to the above inducing steps, wherein when the inducing time is 96h, the protein expression amount tends to steadily and slightly decrease, and the expression is stopped (the protein expression amount trend graph is shown in figure 4).

(2) The initial pH of the induction medium was optimized. Setting the initial pH of the culture medium to 5, 6 and 7, respectively, performing induction expression according to the induction steps, wherein when the induction time is 96h, the protein expression amount tends to steadily and slightly decrease, and the expression is stopped (the protein expression amount trend graph is shown in FIG. 5).

Optimizing according to the conditions to obtain: the optimal induction expression conditions were 28 ℃ at 220rpm, 2% inducer concentration, 7% initial pH of the induction medium, and 72 hours induction time.

Sixthly, purifying and activity measuring of housefly acetylcholinesterase

The target protein was purified by Ni-NTAHis Bind Resin, and the purification effect is shown in FIG. 6. The activity of the expressed housefly acetylcholinesterase is determined by using the acetylcholinesterase activity detection kit, and the enzyme activity can reach 173.3U/mg after purification. The related determination results show that the recombinant housefly acetylcholinesterase can achieve sensitive detection on low-concentration organophosphorus pesticides and carbamate pesticides in vegetable samples, and the detection results are shown in tables 1 and 2.

TABLE 1 inhibition rate of pakchoi samples measured at 1. mu.g/mL and 5. mu.g/mL pesticide concentrations

TABLE 2 inhibition rates of green pepper samples at 1. mu.g/mL and 5. mu.g/mL pesticide concentrations

Attached: the nucleotide and amino acid sequences involved in the present invention are as follows:

SEQ ID NO.1

SEQ ID NO.2

SEQ ID NO.3

although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Sequence listing

<110> Tianjin science and technology university

<120> housefly acetylcholinesterase gene optimized by codon, protein and application

<160>5

<170>SIPOSequenceListing 1.0

<210>1

<211>1737

<212>DNA/RNA

<213> nucleotide sequence of Musca domestica (Unknown)

<400>1

cgcggtctct ttgccacact cgttatactg ttgcgcatgt cagcgctaac gtcagccatg 60

acagatcatc taacggttca aacgaccagc gggccagtac gcggacgttc ggttacagtt 120

cagggtcgcg atgtacacgt ctttaccggc attccgtatg ccaagccgcc cgttgatgat 180

ttgcgttttc gcaaacctgt acctgcagaa ccgtggcatg gtgtcctaga tgcaacacga 240

ctgccagcaa catgcgtaca ggagaggtat gagtacttcc ctggcttttc cggtgaggag 300

atgtggaatc ctaacacaaa tgtatctgaa gattgtttgt ttatgaatat atgggctccg 360

gcgaaggcaa gattacgaca tggtcgtgga accaatggtg gtgagtattc atctaaaacc 420

gatcaggacc atttaataca tagcgcaacg ccacagaaca ccacaaatgg tttgcctata 480

ttaatatgga tttatggcgg tggctttatg actggctcag ccacattgga catttacaac 540

gcagagatta tgtcggccgt gggcaatgtg atcgttgcct cgttccagta tcgactcggt 600

gcatttgggt ttctacatct ttcaccggtt atgccaggtt ttgaagaaga agctcccggc 660

aacgtgggcc tttgggatca ggccatggcc ttgcgttggc ttaaggagaa tgcccgtgca 720

tttggcggca atccggaatg gatgacgctg tttggtgaat cggctggttc gagttccgtg 780

aatgctcaac tgatgtcgcc ggtaacgcgt ggcctggtca aacgtggcat gatgcagtcg 840

gccacaatga atgctccctg gagccacatg acatcagaga aggcggttga aattggtaaa 900

gctttggtaa atgactgtaa ctgtaatgcc tcattgttac cggaaaatcc acgagctgtc 960

atggcttgca tgcgacaggt tgatgcgaaa acaatttctg tccaacaatg gaacccatat 1020

tctggaattt taagttatcc ctcggcccca actatagatg gagcattctt gcccgcagat 1080

ccaatgacac tgttgaaaac agcagatctt agcggttacg atattctgat tggaaatgtt 1140

aaagatgaag gcacatattt tctgctctat gactttattg attatttcga taaggatgat 1200

gcgacatcat tgccacgcga taaatacctg gaaataatga ataatatttt ccaaaaagcc 1260

agtcaagcag aacgagaagc aattatcttc cagtacacaa gttgggaggg taatccaggc 1320

taccagaatc agcaacaaat tggacgagct gtgggtgatc actttttcac ctgtcccaca 1380

aatgaatatg cccaggcatt ggccgaacga ggtgcttcag tgcattatta ttatttcaca 1440

catcgtacca gcacctcatt gtggggcgaa tggatgggtg tcttgcacgg cgatgaaatt 1500

gaatatttct tcggtcagcc attgaacaat tcactgcaat atcgacctgt ggaacgagaa 1560

ttaggcaaac gtatgctcaa ttctgtgatt gaatttgcca aatctggcaa ccctgccgtt 1620

ggtggcgaag aatggcccaa tttctccaag gaagatcccg tttactatgt cttcagtaca 1680

gatgaaaaaa ttgaaaagct acaaagaggt ccattggcca aacgatgctc attctgg 1737

<210>2

<211>579

<212>PRT

<213> amino acids encoded by housefly acetylcholinesterase gene (Unknown)

<400>2

Ala Gly Leu Pro Ala Thr Leu Val Ile Leu Leu Ala Met Ser Ala Leu

1 5 10 15

Thr Ser Ala Met Thr Ala His Leu Thr Val Gly Thr Thr Ser Gly Pro

20 25 30

Val Ala Gly Ala Ser Val Thr Val Gly Gly Ala Ala Val His Val Pro

35 40 45

Thr Gly Ile Pro Thr Ala Leu Pro Pro Val Ala Ala Leu Ala Pro Ala

50 55 60

Leu Pro Val Pro Ala Gly Pro Thr His Gly Val Leu Ala Ala Thr Ala

65 70 75 80

Leu Pro Ala Thr Cys Val Gly Gly Ala Thr Gly Thr Pro Pro Gly Pro

85 90 95

Ser Gly Gly Gly Met Thr Ala Pro Ala Thr Ala Val Ser Gly Ala Cys

100 105 110

Leu Pro Met Ala Ile Thr Ala Pro Ala Leu Ala Ala Leu Ala His Gly

115120 125

Ala Gly Thr Ala Gly Gly Gly Thr Ser Ser Leu Thr Ala Gly Ala His

130 135 140

Leu Ile His Ser Ala Thr Pro Gly Ala Thr Thr Ala Gly Leu Pro Ile

145 150 155 160

Leu Ile Thr Ile Thr Gly Gly Gly Pro Met Thr Gly Ser Ala Thr Leu

165 170 175

Ala Ile Thr Ala Ala Gly Ile Met Ser Ala Val Gly Ala Val Ile Val

180 185 190

Ala Ser Pro Gly Thr Ala Leu Gly Ala Pro Gly Pro Leu His Leu Ser

195 200 205

Pro Val Met Pro Gly Pro Gly Gly Gly Ala Pro Gly Ala Val Gly Leu

210 215 220

Thr Ala Gly Ala Met Ala Leu Ala Thr Leu Leu Gly Ala Ala Ala Ala

225 230 235 240

Pro Gly Gly Ala Pro Gly Thr Met Thr Leu Pro Gly Gly Ser Ala Gly

245 250 255

Ser Ser Ser Val Ala Ala Gly Leu Met Ser Pro Val Thr Ala Gly Leu

260 265 270

Val Leu Ala Gly Met Met Gly Ser Ala Thr Met Ala Ala Pro Thr Ser

275 280285

His Met Thr Ser Gly Leu Ala Val Gly Ile Gly Leu Ala Leu Val Ala

290 295 300

Ala Cys Ala Cys Ala Ala Ser Leu Leu Pro Gly Ala Pro Ala Ala Val

305 310 315 320

Met Ala Cys Met Ala Gly Val Ala Ala Leu Thr Ile Ser Val Gly Gly

325 330 335

Thr Ala Pro Thr Ser Gly Ile Leu Ser Thr Pro Ser Ala Pro Thr Ile

340 345 350

Ala Gly Ala Pro Leu Pro Ala Ala Pro Met Thr Leu Leu Leu Thr Ala

355 360 365

Ala Leu Ser Gly Thr Ala Ile Leu Ile Gly Ala Val Leu Ala Gly Gly

370 375 380

Thr Thr Pro Leu Leu Thr Ala Thr Ile Ala Thr Pro Ala Leu Ala Ala

385 390 395 400

Ala Thr Ser Leu Pro Ala Ala Leu Thr Leu Gly Ile Met Ala Ala Ile

405 410 415

Pro Gly Leu Ala Ser Gly Ala Gly Ala Gly Ala Ile Ile Pro Gly Thr

420 425 430

Thr Ser Thr Gly Gly Ala Pro Gly Thr Gly Ala Gly Gly Gly Ile Gly

435 440445

Ala Ala Val Gly Ala His Pro Pro Thr Cys Pro Thr Ala Gly Thr Ala

450 455 460

Gly Ala Leu Ala Gly Ala Gly Ala Ser Val His Thr Thr Thr Pro Thr

465 470 475 480

His Ala Thr Ser Thr Ser Leu Thr Gly Gly Thr Met Gly Val Leu His

485 490 495

Gly Ala Gly Ile Gly Thr Pro Pro Gly Gly Pro Leu Ala Ala Ser Leu

500 505 510

Gly Thr Ala Pro Val Gly Ala Gly Leu Gly Leu Ala Met Leu Ala Ser

515 520 525

Val Ile Gly Pro Ala Leu Ser Gly Ala Pro Ala Val Gly Gly Gly Gly

530 535 540

Thr Pro Ala Pro Ser Leu Gly Ala Pro Val Thr Thr Val Pro Ser Thr

545 550 555 560

Ala Gly Leu Ile Gly Leu Leu Gly Ala Gly Pro Leu Ala Leu Ala Cys

565 570 575

Ser Pro Thr

<210>3

<211>1737

<212>DNA/RNA

<213> nucleotide sequence of housefly acetylcholinesterase gene (Unknown)

<400>3

cgaggtctct ttgccacact cgttatcctg ttgcgaatgt cagctctaac gtcagccatg 60

acagatcatc taacggttca aacgaccagc gggccagtac gaggacgttc ggttacagtt 120

cagggtcgag atgtacacgt ctttaccggc attccatatg ccaagccacc cgttgatgat 180

ttgcgttttc gaaaacctgt acctgcagaa ccatggcatg gtgtcctaga tgcaacacga 240

ctgccagcaa catgtgtaca ggagaggtat gagtacttcc ctggcttttc cggtgaggag 300

atgtggaatc ctaacacaaa tgtatctgaa gattgtttgt ttatgaatat ctgggctcca 360

gctaaggcaa gattacgaca tggtcgtgga accaatggtg gtgagtattc atctaaaacc 420

gatcaggacc atttaatcca tagcgcaacg ccacagaaca ccacaaatgg tttgcctatc 480

ttaatctgga tttatggcgg tggctttatg actggctcag ccacattgga catttacaac 540

gcagagatta tgtcggccgt gggcaatgtg atcgttgcct cgttccagta tcgactcggt 600

gcatttgggt ttctacatct ttcaccagtt atgccaggtt ttgaagaaga agctcccggc 660

aacgtgggcc tttgggatca ggccatggcc ttgcgttggc ttaaggagaa tgcccgtgca 720

tttggcggca atccagaatg gatgacgctg tttggtgaat cggctggttc gagttccgtg 780

aatgctcaac tgatgtcgcc agtaacgcgt ggcctggtca aacgtggcat gatgcagtcg 840

gccacaatga atgctccctg gagccacatg acatcagaga aggctgttga aattggtaaa 900

gctttggtaa atgactgtaa ctgtaatgcc tcattgttac cagaaaatcc acgagctgtc 960

atggcttgta tgcgacaggt tgatgctaaa acaatttctg tccaacaatg gaacccatat 1020

tctggaattt taagttatcc ctcggcccca actatcgatg gagcattctt gcccgcagat 1080

ccaatgacac tgttgaaaac agcagatctt agcggttacg atattctgat tggaaatgtt 1140

aaagatgaag gcacatattt tctgctctat gactttattg attatttcga taaggatgat 1200

gctacatcat tgccacgaga taaatacctg gaaatcatga ataatatttt ccaaaaagcc 1260

agtcaagcag aacgagaagc aattatcttc cagtacacaa gttgggaggg taatccaggc 1320

taccagaatc agcaacaaat tggacgagct gtgggtgatc actttttcac ctgtcccaca 1380

aatgaatatg cccaggcatt ggccgaacga ggtgcttcag tgcattatta ttatttcaca 1440

catcgtacca gcacctcatt gtggggcgaa tggatgggtg tcttgcacgg cgatgaaatt 1500

gaatatttct tcggtcagcc attgaacaat tcactgcaat atcgacctgt ggaacgagaa 1560

ttaggcaaac gtatgctcaa ttctgtgatt gaatttgcca aatctggcaa ccctgccgtt 1620

ggtggcgaag aatggcccaa tttctccaag gaagatcccg tttactatgt cttcagtaca 1680

gatgaaaaaa ttgaaaagct acaaagaggt ccattggcca aacgatgttc attctgg 1737

<210>4

<211>21

<212>DNA/RNA

<213> pPIC9K-His plasmid primers: s2(Unknown)

<400>4

gactggttcc aattgacaag c 21

<210>5

<211>21

<212>DNA/RNA

<213> pPIC9K-His plasmid primers: a2(Unknown)

<400>5

gcaaatggca ttctgacatc c 21

Claims

1. A housefly acetylcholinesterase gene optimized by codon is characterized in that: the nucleotide sequence of the housefly acetylcholinesterase gene is SEQ ID NO. 3.

2. The codon-optimized housefly acetylcholinesterase gene according to claim 1, wherein: the housefly acetylcholinesterase gene is subjected to codon optimization on the basis of the existing housefly acetylcholinesterase gene, and can encode protein with acetylcholinesterase activity.

3. The codon optimized housefly acetylcholinesterase gene according to claim 1 or 2, wherein: the housefly acetylcholinesterase gene is obtained by optimizing 29 codons on the basis of an original gene sequence SEQ ID NO.1, namely ccg → cca cgc, cgg → cga gcg → gct tgc → tgt ata → atc.

4. The codon-optimized housefly acetylcholinesterase gene-encoded amino acid of any one of claims 1 to 3, wherein: the sequence of the amino acid is SEQ ID NO. 2.

5. An expression vector comprising the housefly acetylcholinesterase gene according to any one of claims 1 to 3.

6. A eukaryotic expression cell comprising the expression vector of claim 5.

7. A method for producing a protein expressed by the codon-optimized housefly acetylcholinesterase gene according to any one of claims 1 to 3.

8. The method of claim 7, wherein: the method comprises the following steps:

⑴ obtaining optimized gene sequence by gene synthesis;

9. Use of a protein expressed by a codon-optimized housefly acetylcholinesterase gene according to any of claims 1 to 3 for the detection of pesticides.

10. Use of a protein expressed by a codon-optimized housefly acetylcholinesterase gene according to any of claims 1 to 3.