CN104865248B - A kind of preparation method for the recombinant silkworm acetylcholinesterase for being used to detect pesticide residue - Google Patents
A kind of preparation method for the recombinant silkworm acetylcholinesterase for being used to detect pesticide residue Download PDFInfo
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Abstract
The invention discloses a kind of preparation method for the recombinant silkworm acetylcholinesterase for being used to detect pesticide residue.The present invention is by silkworm acetylcholinesterasegene gene and anchorin geneAGα1It is connected to be inserted into Yeast expression carrier pPIC9K and is built into surface display vector and converts Pichia pastoris GS115, screen positive recombinant, and induced expression is carried out to positive recombinant using methanol, centrifugation removes supernatant after expression, and thalline is freeze-dried up to recombinant silkworm acetylcholinesterase.The restructuring enzyme stability that is prepared is good, easy to operate, detection limit is low, high sensitivity, especially of low cost, Site Detection can be carried out in family, market, vegetables production base etc., the examination of a large amount of samples is very suitable for, there is important practical application dissemination.
Description
Technical field
The present invention relates to Fast Determination of Pesticide Residue technical field, and in particular, to a kind of to be used to detect pesticide residue
The preparation method of recombinant silkworm acetylcholinesterase, is used to detect Organophosphorus and carbamate pesticides class more particularly, to one kind
The preparation method of the recombinant silkworm acetylcholinesterase of pesticide residue.
Background technology
The use of pesticide also causes environment with product serious while great economic benefit is brought to agricultural production
Pollution, very big influence is brought to people's physical and mental health.The method of Detecting Pesticide mainly has instrumental method at present, is immunized
Analytic approach and enzyme inhibition etc..Instrumental method detection sensitivity is high, selectivity is good, qualitative, quantitative can be carried out at the same time, result is accurate
It is true reliable, it is standard method and the referee method of current detection pesticide residue, but instrumental method is needed using the big of laboratory costliness
Type analysis instrument, analysis work is completed by professional technician, and sample pre-treatments are cumbersome, the time is longer, thus
Laboratory is can be only applied to, it is difficult to adapt to the requirement of the simple and quick and a large amount of sample pesticide residue analysis in scene, is unfavorable in base
Layer is promoted and applied.Immunoassay has strong specificity, high sensitivity, quick, simple operation and other advantages, but this method pair
The high selectivity of reagent, is easily influenced, the compound similar to structure also has certain intersection anti-be subject to detection sample substrate
Should, and the preparation of the wide spectrum specific antibody with more residual identifications is still in conceptual phase.Current China pesticide residue is quickly examined
The development time of enzyme inhibition is long in survey technology, more ripe compared with other fast determining methods, and operation is simpler, and cost is more
Low, it is quick, sensitive, accurate to have the advantages that, can effectively make up that instrument analytical method pre-treatment is cumbersome, testing cost is high, it is difficult to
Meet the deficiency of high throughput, quick and on-line checking etc., be more suitable for the national conditions in China, can be as instrument analytical method
Useful supplement.But commercially available Fast Determination of Pesticide Residue kit or test strips its core material cholinesterases are mostly direct
Obtained from extractions such as housefly, horse serum, structure of fish muscle, pork liver, sheeps blood erythrocyte, plant tissues, these are straight from organism
The cholinesterase of separation and Extraction is connect there are the deficiencies of extracted amount is low, sensitivity difference is big, stability is poor between purification difficult, species,
Therefore easily occurs false positive in actually detected, shortcoming is more in.Wang Dong etc. divides housefly acetylcholinesterasegene gene
It is not building up in protokaryon and eukaryotic expression system, protokaryon and eukaryotic expression is carried out respectively, still, it turns out that prokaryotic expression comes out
Restructuring acetylcholine esterase of housefly vigor it is very low, and eukaryotic expression come out restructuring acetylcholine esterase of housefly without activity.
The content of the invention
The present invention is in order to overcome the above-mentioned deficiency of the prior art, there is provided one kind is used to detect Organophosphorus and carbamate pesticides class
The preparation method of the recombinant silkworm acetylcholinesterase of pesticide residue.The recombinant silkworm acetylcholine being prepared by this method
Esterase yield is big, high sensitivity and stabilization, and can be used for quickly detecting at scenes such as family, market, vegetables production bases
The residual of machine phosphorus and carbamate chemicals for agriculture.
It is a further object to provide a kind of enzyme preparation for detecting Organophosphorus and carbamate pesticides pesticide residue.
To achieve these goals, the present invention is achieved by following scheme：
A kind of preparation for the recombinant silkworm acetylcholinesterase for being used to detect Organophosphorus and carbamate pesticides pesticide residue
Method, comprises the following steps：By silkworm acetylcholinesterasegene gene and anchorin geneAGα1It is connected and is inserted into Yeast expression
Surface display vector is built into carrier pPIC9K and converts Pichia pastoris GS115, screens positive recombinant, and utilize methanol pair
Positive recombinant carries out induced expression, and centrifugation removal supernatant, thalline is freeze-dried up to recombinant silkworm second after expression
Acetylcholinesterase.
Also someone is using recombination and expression techniques come Prepare restructuring acetylcholinesterase in the prior art, and such as Wang Dong is by housefly
Acetylcholinesterasegene gene is building up in protokaryon and eukaryotic expression system respectively, carries out protokaryon and eukaryotic expression respectively, still, knot
Fruit finds that the restructuring acetylcholine esterase of housefly vigor that prokaryotic expression comes out is very low, and the restructuring housefly acetyl that eukaryotic expression comes out
Cholinesterase is without activity.The present invention is by silkworm acetylcholinesterasegene gene and anchorin geneAGα1It is connected and is building up to surface
In display carrier and Pichia pastoris is converted, the activity of the recombinase obtained by immobilization display technique is very well.
Preferably, the nucleotide sequence of the silkworm acetylcholinesterasegene gene such as SEQ ID NO：Shown in 1.
The recombinant silkworm acetylcholinesterase being prepared by as above method.
As above the recombinant silkworm acetylcholinesterase that method is prepared is in detection Organophosphorus and carbamate pesticides class pesticide
Application in residual.
A kind of enzyme preparation for being used to detect Organophosphorus and carbamate pesticides pesticide residue, including restructuring man as described above
Silkworm acetylcholinesterase, auxiliary reagent A, auxiliary reagent B and auxiliary reagent C；Wherein, the auxiliary reagent A is acetic acid-α-naphthalene
Ester, auxiliary reagent B are solid indigo plant B salt, and auxiliary reagent C is the phosphate buffer pulvis of pH 7.5.
Preferably, the auxiliary reagent A using when be dissolved in acetone, be made into concentration be 0.03 mol/L solution.
Preferably, the auxiliary reagent B using when be dissolved in methanol, be made into concentration be 0.06 mol/L solution.
Preferably, the concentration when auxiliary reagent C is used is 50mM.
The preparation of auxiliary reagent A：Ester acetic acid-α-naphthylacetate is dissolved in organic solvent, organic solvent is acetone, stirring
It is 0.03 mol/L uniformly to make its concentration, and solution can preserve one month in 4 DEG C.
The preparation of auxiliary reagent B：Color developing agent is consolidated into blue B salt and is dissolved in organic solvent, organic solvent is methanol, and stirring evenly makes
Its concentration is 0.06 mol/L, and stability is better than its aqueous solution.
The preparation of auxiliary reagent C：By Na2HPO412H2O and NaH2PO42H2O in mass ratio 10:1 mixes, and the used time is molten
In a certain amount of distilled water, its concentration is 50mM, pH 7.5.
The enzyme preparation of the present invention can also use in detection process and arrive standard color comparison card, and standard color comparison card is according to various concentrations
Pesticide shown color is made when detecting.
The present invention application principle be：Under normal circumstances, acetylcholinesterase can be catalyzed nerve conduction metabolite second
The substitute acetic acid of phatidylcholine-α-naphthylacetate hydrolysis, its hydrolysate alpha-Naphthol can consolidate indigo plant B reactant salts with color developing agent, and generation is purplish red
The azo-compound of color.And Organophosphorus and carbamate pesticides class pesticide has inhibitory action to acetylcholinesterase normal function, make
Catalysis, hydrolysis, the process of discoloration change, and in the presence of having pesticide, the color of solution is in light red or the original Huang of solid indigo plant B salt
Color, rather than aubergine, the organophosphor or carbamates for whether having high dose in sample are can determine whether out according to the change of color
The presence of pesticide.
A kind of method for detecting Organophosphorus and carbamate pesticides pesticide residue, comprises the following steps, by recombinant silkworm second
Acetylcholinesterase obtains liquid enzyme formulation after being mixed with auxiliary reagent C, sample to be tested and appropriate amount of fluid enzyme preparation is mixed, 2 ~ 3 points
Auxiliary reagent A and auxiliary reagent B is added after clock, the color of prepare liquid is observed after 1 ~ 2 minute, if there is aubergine, is then illustrated
Pesticide residue is not exceeded, if there is light red or yellow, then illustrates that pesticide residue is exceeded；In addition, by testing result and standard
Colorimetric card is made comparisons, then can know the residual quantity of pesticide roughly.
The container used during detection can be the forms such as centrifuge tube, teat glass, blood bag, be equipped with colorimeter, can also be with
Speed is surveyed meter form and is used.
The present invention can both detect the residual of organophosphor or carbamate chemicals for agriculture in vegetables, can also detect fruit,
The residual of organophosphor or carbamate chemicals for agriculture in underground water, grain.
Compared with prior art, the invention has the advantages that：
（1）Stability is good：Recombinant silkworm acetylcholinesterase is to be fixed on saccharomycete using what yeast surface display obtained
The immobilised enzymes in body surface face, is easy to preserve after freeze-dried, and it is still very high that 2 years vigor are placed under the conditions of low-temperature dark；Auxiliary examination
Agent is solid-state form, and storage life is greatly prolonged than the general solution form term of validity.
（2）High sensitivity：Extremely low to organophosphorus pesticide and carbamate chemicals for agriculture test limit, general detection is limited to several
Ppb, minimum reachable 0.2 ppb.
（3）It is time-consuming short：General operation person can once handle multiple samples at the same time, and average measurement one is completed from measure is sampled to
A sample only needs 3 ~ 5 minutes.
（4）It is easy to operate：Expensive large-sized analytic instrument need not be used, professional technician's analysis, side is also not required
Just it is detected at scenes such as family, the market of farm produce, Vegetable Bases.
（5）It is of low cost：After recombinant silkworm acetylcholinesterase Yeast expression engineered strain structure is completed, using first
The a large amount of induced expressions of alcohol, yield is high and cheap, and auxiliary reagent expense almost can be ignored.
Brief description of the drawings
Fig. 1 is the structure flow chart of recombinant silkworm acetylcholinesterase surface display vector.
Fig. 2 is common organophosphorus pesticide structure diagram.
Fig. 3 is conventional amino formate ester pesticide structure schematic diagram.
Fig. 4 is suppression curve of several organophosphorus pesticides to recombinant silkworm acetylcholinesterase.
Fig. 5 is suppression curve of several carbamates chemicals for agriculture to recombinant silkworm acetylcholinesterase.
Embodiment
The present invention is made with reference to Figure of description and specific embodiment and further being elaborated, the embodiment
It is served only for explaining the present invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is such as without spy
Different explanation, is conventional method；Used material, reagent etc., unless otherwise specified, for the reagent commercially obtained
And material.
Embodiment 1
By silkworm acetylcholinesterasegene gene and anchorin geneAGα1It is connected and is inserted into Yeast expression carrier pPIC9K
In be built into surface display vector, whole vector construction process is as shown in Figure 1.The surface display vector conversion Pichia pastoris built up
GS115, screens positive recombinant.
Comprise the following steps that：
1st, using Trizol reagents extracting silkworm head total serum IgE；With RT-PCR kit using silkworm head total serum IgE as mould
Plate, Oligo (dT)18For primer, reverse transcription synthesis the first chains of cDNA under the action of M-MuLV reverse transcriptase；With the first chain cDNA
Pass through PCR amplification silkworm acetylcholinesterasegene gene for templatebmace（Full length gene sequence such as SEQ ID NO.1）, willbmace
Gene is connected into PMD-18-T carriers, connection product conversion conversion bacillus coli DH 5 alpha；With recombinant plasmid PMD-18-T-bmaceFor
Template PCR amplifications bandMluI restriction enzyme sitebmaceGene, and introduce 6 × His labels at 5 ' ends.
2nd, saccharomyces cerevisiae INVSc1 genomic DNAs are extracted using Yeast genome extraction agent box, passed through as template
PCR amplification anchorin geneAGα1（AGα1Gene complete sequence GenBank sequence numbers：X16861.1）, while in amplified fragments
5 ' end introduce one section of linker sequence（Linker sequences are shown in Table 1）.
3rd, by PCR by step 1bmaceGene and the anchorin gene in step 2AGα1It is pre-splicing to be connected together,
Fusion is expanded by Overlap extension PCRbmace-AGα1。
4th, by the fusion in Yeast expression carrier pPIC9K and stepbmace-AGα1Do double digestion at the same time（MluⅠ、NotⅠ）, digestion is later by the two composition recombinant plasmid pPIC9K- that links togetherbmace-AGα1, connection product conversion large intestine
Bacillus DH5 α.By recombinant vector pPIC9K- after PCR identifications and digestion identification are correctbmace-AGα1Convert Pichia pastoris
GS115 forms transformant GS115/pPIC9K-bmace-AGα1.Identify that correct integration transformation of target gene is restructuring through PCR
Son, the induced expression available for next step.
All primers and sequence used in 1. PCR amplification of table
Embodiment 2
The preparation of recombinant silkworm acetylcholinesterase：By Yeast expression engineered strain GS115/pPIC9K-bmace-AGα1
Induced expression 4 days, centrifugation remove supernatant, after thalline is freeze-dried, up to recombinant silkworm acetylcholinesterase, are sub-packed in modeling
Expect in centrifuge tube, buckle closure preserves under the conditions of being placed in low-temperature dark.Used time takes the solid polypeptide formulation that 0.1 g is freeze-dried, and adds 50
ML auxiliary reagent C, vibration are uniformly mixed and liquid enzyme formulation are made.
Recombinant silkworm Acetylcholinesterasein measures：
800 μ L of recombinant silkworm acetylcholinesterase are taken, are separately added into 100 μ L substrates and 100 μ L color developing agents reaction 5
Min, while the body with the bacteria suspension of empty bacterium GS115/pPIC9K instead of the recombinant silkworm acetylcholinesterase suspension of surface display
System is used as blank control, and the absorbance under supernatant 412 nm wavelength of measure is collected by centrifugation, is defined when calculating enzyme activity in 1 minute
The enzyme amount that 1 micromole substrate can be catalyzed is a unit.It is computed, the thalline enzyme activity of induced expression reaches 104.2 U/mL, presses
It is then 787.7 U/g that thalline weight, which calculates,.
In order to verify the active situation of the recombinant silkworm acetylcholinesterase of preparation, several organophosphorus pesticides and several are determined
Inhibition of the carbamate chemicals for agriculture to recombinant silkworm acetylcholinesterase is planted, suppression curve is shown in Fig. 4 and Fig. 5 respectively.
The preparation of auxiliary reagent A：Weigh 56 mg acetic acid-α-naphthylacetate to be dissolved in 10 mL acetone, that is, be configured to 0.03
Mol/L acetic acid-α-naphthylacetate acetone soln, 4 DEG C can preserve one month.
The preparation of auxiliary reagent B：Weigh 285 mg and consolidate indigo plant B salt and be dissolved in 10 mL methanol, that is, be configured to 0.06 mol/L
Gu indigo plant B salt methanol solutions, using effect is preferable in 2 weeks.If without methanol, it can be configured with distilled water, but solid indigo plant B saline solutions are not
Such as solid indigo plant B salt methanol solution stability is good, it is necessary to prepare same day use, is otherwise easy to from initial that faint yellow to be changed into depth brown
Color, influences the judgement of result.
The preparation of auxiliary reagent C：By Na2HPO412H2O and NaH2PO42H2O in mass ratio 10:1 mixes, and the used time is molten
In a certain amount of distilled water, its concentration is 50mM, pH 7.5.
Embodiment 3
The remaining application of pesticide in each sample is detected of enzyme preparation described in embodiment 2.
Fruit：A little fruit is shredded, takes about 2 g fruit pieces to be placed in 50 mL conical flasks, adds 10 mL auxiliary examinations
Agent C, vibrates 1 minute, static.An empty centrifuge tube is taken to add 100 μ L restructuring enzyme preparations and 2.5 mL extract from fruit, mixing
After being uniformly incubated 2 minutes, each 100 μ L of auxiliary reagent A and auxiliary reagent B are added, are developed the color 2 ~ 3 minutes, observe liquid in centrifuge tube
Color.If there is aubergine, then illustrate that pesticide residues in fruits is not exceeded, if there is light red or yellow, then illustrate
Pesticide residues in fruits is exceeded.Make comparisons with standard color comparison card, then can know the residual quantity of pesticide roughly.
Vegetables：Vegetable surface soil is rinsed out, is cut into 1 cm or so square fragments, takes about 2 g of vegetable sample to be placed in 50
In mL conical flasks, 10 mL auxiliary reagent C are added, are vibrated 1 minute, it is static.An empty centrifuge tube is taken to add 100 μ L recombinases
Preparation and 2.5 mL vegetables extracting solutions, are uniformly mixed after being incubated 2 minutes, add each 100 μ L of auxiliary reagent A and auxiliary reagent B,
Colour developing 2 ~ 3 minutes, observes the color of liquid in centrifuge tube.If there is aubergine, then illustrate that Pesticide Residues in Vegetables is not exceeded,
If there is light red or yellow, then illustrate that Pesticide Residues in Vegetables is exceeded.Make comparisons, then can know roughly with standard color comparison card
The residual quantity of pesticide.
Drinking Water：An empty centrifuge tube is taken to add 100 μ L restructuring enzyme preparations and 2.5 mL Drinking Waters, mixing
After being uniformly incubated 2 minutes, each 100 μ L of auxiliary reagent A and auxiliary reagent B are added, are developed the color 2 ~ 3 minutes, observe liquid in centrifuge tube
Color.If there is aubergine, then illustrate that pesticide residue is not exceeded in Drinking Water, if there is light red or yellow,
Then illustrate that pesticide residue is exceeded in Drinking Water.Make comparisons with standard color comparison card, then can know the residual quantity of pesticide roughly.
Grain：Take about 2 g grains to be placed in 50 mL conical flasks, add 10 mL auxiliary reagent C, vibrate 1 minute, it is quiet
Only.Take an empty centrifuge tube to add 100 μ L restructuring enzyme preparations and 2.5 mL grain extracting solutions, be uniformly mixed after being incubated 2 minutes,
Each 100 μ L of auxiliary reagent A and auxiliary reagent B are added, are developed the color 2 ~ 3 minutes, observe the color of liquid in centrifuge tube.If there is
Aubergine, then illustrate that Pesticide Residues In Grain is not exceeded, if there is light red or yellow, then illustrates that Pesticide Residues In Grain surpasses
Mark.Make comparisons with standard color comparison card, then can know the residual quantity of pesticide roughly.
SEQUENCE LISTING
<110>Agricultural University Of South China
<120>A kind of preparation method for the recombinant silkworm acetylcholinesterase for being used to detect pesticide residue
<130>
<160> 14
<170> PatentIn version 3.3
<210> 1
<211> 1914
<212> DNA
<213>Silkworm bmace full length gene sequences
<400> 1
atgatcaact acggcaagat tgtattcact aagcttcttc tatgcgtact catgtccggt 60
acttttgctc gatcttgggc caatcaccat gataccacaa catctacaac acaaactacc 120
ccaacgacaa gtccggtacc aaaaaatatc cacaacgatc cacttattgt cgaaacaaag 180
agcggtctca tcaaaggata cgcaaaaact gtaatgggac gcgaggtaca catttttacg 240
ggtatcccgt ttgcgaaacc tccattagga cccctgagat tccgtaaacc ggtaccaatc 300
gagccatggc atggcgtgct tgaagcaaac ttaatgccaa acagttgcta tcaagaacgc 360
tacgaatatt tcccaggttt cgaaggagaa gaaatgtgga atccaaatac taatatatca 420
gaagattgcc tttatttgaa tatttgggta ccacagcact tacgagttcg tcaccatcaa 480
gataaaccac tcgccgaaag acctaaagtg ccgattcttg tgtggattta cggcggtggc 540
tacatgagtg gcgcggctac acttgaccta tataaagcag atataatggc atctacaagc 600
gacgtaatag tggcttctat gcaatacagg gttggtgcat ttggattttt atatttgaat 660
aaatattttt ctccgggtag tgaagaagct cctggaaata tgggtttatg ggatcaacaa 720
ctcgctattc gttggataaa agagaacgct cgtgcttttg gaggagaccc tgaactcatt 780
acgctgttcg gggaatctgc cggtggcggt agtgtaagcc ttcatatgct atcacctgaa 840
atgaaaggat tgtttaaaag aggtatattg caatcaggaa cgttgaatgc accttggagt 900
tggatgactg gagaaagagc tcaagatatt ggaaaagtat taattgatga ctgtaactgc 960
aacagtagtc ttttagccaa ggatcctagt ctcgtaatgg attgcatgcg tggagttgac 1020
gctaaaacga tttctgtcca gcaatggaat tcttatactg gaattttggg ttttccgtcc 1080
gcacctacgg ttgatggtat ttttttgcca aaagatcctg ataccatgat gaaggaagga 1140
aatttccata atagtgaagt gctacttggc agtaaccaag acgaagggac atattttttg 1200
ctgtacgact tcctggatta tttcgaaaag gatgggccta gttttcttca gagggagaaa 1260
tttctcgaaa tcgttgacac tattttcaag gacttttcta aaattaaaag agaagccatt 1320
gtgttccagt atacagattg ggaagagatc accgacggat atttgaacca gaagatgata 1380
gctgatgtcg taggagacta cttcttcgta tgccccacta actacttcgc cgaaatactt 1440
gccgacgctg gtgtcgatgt ttactattac tattttactc atcgtaccag cacaagtctc 1500
tggggagaat ggatgggcgt gatgcatggt gacgaaatgg aatatgtttt tggacatccc 1560
ttgaacatgt cccttcagta ccattcccgg gagcgtgatt tagcagcaca cattatgcag 1620
tctttcacac agtttgctct taccggaaaa cctcacaagc ctgacgagaa gtggcctctg 1680
tactcccggt cttcgcctca ttactacaca tacacggcgg tgggtccaag cggtccagct 1740
ggaccccgcg gcccgcgtgc ctccgcttgc gctttctgga acgatttctt gaacaaactt 1800
aacgagttgg agcgtgtacc gtgtgacggc gccgtgaccg gtccttacag cagtgtcgcc 1860
ggcactgccc tccctgtaac ccttctcacc actttggcaa tcactattgc tttg 1914
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<213> bmace（F）
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cgacgcgtca tcatcatcat catcatagat cttgggctaa ccaccacga 49
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<213> bmace（R）
<400> 3
gcggcggccg cagaggagta tggaccggta aca 33
<210> 4
<211> 51
<212> DNA
<213> bmace -Flag（F）
<400> 4
gcgacgcgtg actacaagga cgacgacgac aagatgatca actacggtaa g 51
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<211> 32
<212> DNA
<213> AGα1-linker（F）
<400> 5
gccgcggccg cgaatagcag gtacgacaaa ag 32
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<211> 49
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<213> AGα1（R）
<400> 6
aggcggaggt ggctctggcg gtggcggatc gaacctcggt acagctagc 49
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<213> ace-FQtest（F）
<400> 7
gcatcaagaa cgctacgaat 20
<210> 8
<211> 21
<212> DNA
<213> ace-FQtest（R）
<400> 8
cgccctgtat tgcatagaag c 21
<210> 9
<211> 20
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cgtcggtatt aacggtttcg 20
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<213> GAP（R）
<400> 10
gcttgtaagc cttgtgggt 19
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<211> 50
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<213> ace-linker（R）
<400> 11
ccagagccac ctccgcctga accgcctcca ccagaggagt atggaccggt 50
<210> 12
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<213> α-factor
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tactattgcc agcattgctg c 21
<210> 13
<211> 21
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<213> AOX(F)
<400> 13
gactggttcc aattgacaag c 21
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gcaaatggca ttctgacatc c 21
Claims (8)
- A kind of 1. preparation side for the recombinant silkworm acetylcholinesterase for being used to detect Organophosphorus and carbamate pesticides pesticide residue Method, it is characterised in that comprise the following steps：1）Using Trizol reagents extracting silkworm head total serum IgE；With RT-PCR kit with Silkworm head total serum IgE is template, and Oligo (dT) 18 is primer, the reverse transcription synthesis cDNA under the action of M-MuLV reverse transcriptase First chain；It is template by PCR amplification silkworm acetylcholinesterasegene gene bmace using the first chain cDNA, bmace genes is connected into PMD-18-T carriers, connection product conversion conversion bacillus coli DH 5 alpha；Expand by template PCR of recombinant plasmid PMD-18-T-bmace Increase the bmace genes with I restriction enzyme sites of Mlu, and 6 × His labels are introduced at 5 ' ends；2）Saccharomyces cerevisiae INVSc1 genomic DNAs are extracted using Yeast genome extraction agent box, are expanded as template by PCR Increase anchorin Gene A/G α 1, while one section of linker sequence is introduced at 5 ' ends of amplified fragments；3）By PCR by step 1）In bmace genes and step 2）In anchorin Gene A/G α 1 it is pre-splicing be connected together, lead to Lap over extension PCR amplification fusion bmace-AG α 1；4）Fusion bmace-AG α 1 in Yeast expression carrier pPIC9K and step are done into double digestion at the same time, digestion later will The two, which links together, forms recombinant plasmid pPIC9K-bmace-AG α 1, connection product conversion bacillus coli DH 5 alpha；By PCR Recombinant vector pPIC9K-bmace-AG α 1 are converted into Pichia pastoris GS115 after identification and digestion identification are correct and form transformant GS115/pPIC9K-bmace-AG α 1, screen positive recombinant, and carry out induced expression, table to positive recombinant using methanol Centrifugation removes supernatant after reaching, and thalline is freeze-dried up to recombinant silkworm acetylcholinesterase；The primer of the PCR amplification is as shown in SEQ ID NO.2~SEQ ID NO.14.
- 2. the recombinant silkworm acetylcholinesterase that method is prepared according to claim 1.
- 3. recombinant silkworm acetylcholinesterase is in detection Organophosphorus and carbamate pesticides pesticide residue according to claim 2 In application.
- 4. a kind of enzyme preparation for being used to detect Organophosphorus and carbamate pesticides pesticide residue, it is characterised in that will including right Seek recombinant silkworm acetylcholinesterase, auxiliary reagent A, auxiliary reagent B and the auxiliary reagent C described in 2；Wherein, the auxiliary examination Agent A is acetic acid-α-naphthylacetate, and auxiliary reagent B is solid indigo plant B salt, and auxiliary reagent C is the phosphate buffer pulvis of pH 7.5.
- 5. enzyme preparation according to claim 4, it is characterised in that the auxiliary reagent A using when be dissolved in acetone, be made into Concentration is the solution of 0.03 mol/L.
- 6. enzyme preparation according to claim 4, it is characterised in that the auxiliary reagent B using when be dissolved in methanol, be made into Concentration is the solution of 0.06 mol/L.
- 7. enzyme preparation according to claim 4, it is characterised in that the concentration when auxiliary reagent C is used is 50mM.
- A kind of 8. method for detecting Organophosphorus and carbamate pesticides pesticide residue, it is characterised in that by described in claim 4 Recombinant silkworm acetylcholinesterase obtains liquid enzyme formulation after being mixed with auxiliary reagent C, by sample to be tested and appropriate amount of fluid enzyme preparation Mixing, adds auxiliary reagent A and auxiliary reagent B described in claim 4, prepare liquid is observed after 1~2 minute after 2~3 minutes Color, if there is aubergine, then illustrates that pesticide residue is not exceeded, if there is light red or yellow, then illustrates pesticide residue It is exceeded；In addition, testing result is made comparisons with standard color comparison card, then the residual quantity of pesticide can be known roughly.
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| CN101178400A (en) * | 2007-11-20 | 2008-05-14 | 苏州大学 | Method for inspecting wild mulberry silkworm having organophosphorus pesticide resistance species |
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| CN101178400A (en) * | 2007-11-20 | 2008-05-14 | 苏州大学 | Method for inspecting wild mulberry silkworm having organophosphorus pesticide resistance species |
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| 家蚕乙酰胆碱酯酶基因 bmace 在毕赤酵母GS115 中的表达及其分析;何永盛等;《中国农业科学》;20121231;第45卷(第11期);第2190页右栏最后1段-第2194页左栏最后1段 * |
| 酿酒酵母表面展示表达系统及应用;郭钦等;《中国生物工程杂志》;20081215;第28卷(第12期);第116-122页 * |
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