CN108588084A - A kind of genetic engineering horseshoe crab blood G-factor and its preparation method and application - Google Patents
A kind of genetic engineering horseshoe crab blood G-factor and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of genetic engineering horseshoe crab blood G-factors and its preparation method and application.The present invention has carried out codon optimization to the nucleotide sequence of G-factor α subunits and beta subunit gene, and sequence is respectively such as SEQ ID NO after optimization：1 and SEQ ID NO：Shown in 2, while the prokaryotic expression carrier of G-factor gene subunit optimization is successfully constructed, is transformed into Escherichia coli and carries out efficient induced expression, extraction and purifying expression albumen, then effective integration is carried out to two subunits；Bioactivity verification finally has been carried out to the destination protein after fusion.The present invention successfully obtains can be in the α subunits and β subunits of E. coli, and two subunits are re-assemblied into the Viability and higher G-factor of purity, its preparation process is simple, it is at low cost, with good biological function, the reagents for producing detection fungal infection is can be used to, false positive can be reduced, greatly reduce the demand to wild horseshoe crab resource simultaneously, has a extensive future.
Description
Technical field
The invention belongs to biomedicine fields.More particularly, to a kind of genetic engineering horseshoe crab blood G-factor and preparation method thereof
And application.
Background technology
Deep fungal infection incidence rises year by year, and because lacking effective early diagnosis means, case fatality rate is high.
Currently, the method for detection fungal infection has the biopsy of blood culture tissue, round pcr and immunological method.Wherein, blood culture and group
Knit biopsy because cultural method time-consuming with detection positive rate it is low, should not early diagnose；PCR can only detect known pathomycete sense
Dye is not suitable for the rare opportunistic fungus infection of early diagnosis；And immunological method will do a variety of fungal antigen screenings, leakiness
It examines, and time-consuming and uneconomical.The severity of deep fungal infection often with blood plasma (1-3)-beta-D- glucans（That is (1-3)-
Callose）Elevated levels it is consistent, and (1-3)-callose can specific activation from the G-factor of limulus blood cell, this
Process is known as G experiments.G experiments have in the early diagnosis of deep fungal infection compared with hypersensitivity and specificity, so clinical
Examine the method for early diagnosis for having included G experiments as fungal infection.
But because horseshoe crab coagulation process has two different activated pathway, one be endotaxin mediate C factor approach：C because
Son（FC）It is activated in conjunction with endotoxin, then activates Factor B, the Factor B of activation is by proclotting enzyme（Proclotting
enzyme）It is converted into coagulase（clotting enzyme）, coagulase converts coagulagen to coagulated protein, solidifies egg
White be cross-linked with each other is dehydrated and forms gel；Another is the G-factor mediated by 1,3- calloses（FG）Approach, equally can be with
Cause similar agglutinating reaction, generate false positive results, therefore, reagents cannot be directly used to detection fungal infection.Currently, to the greatest extent
Pipe has increases the method for detection specificity in reagents by adding anti-grease polysaccharide material, but in it can not block completely
The interference that toxin tests G.In addition, reagents can only prepare reagents heavy dependence horseshoe crab money by collecting the production of horseshoe crab hemolymph
Source, such as with biological products, medicine：Injection medicament, chemical drugs category, radiopharmaceutical, antibiotics, vaccine, dialysis
The preparations such as liquid and medical instrument（Such as disposable syringe, implantable biomaterial）Equal production units increase rapidly, fungi sense
The demand for contaminating detection reagent is increasing so that the quantity of horseshoe crab gradually decreases in ocean.
Therefore, not against horseshoe crab hemolymph is used, the horseshoe crab blood G-factor for diagnosing fungal infection becomes increasingly to weigh for exploitation
It wants.
Invention content
The purpose of the invention is to overcome the deficiencies of the prior art and provide it is a kind of can be in E. coli
α subunit genes and beta subunit gene, the subunit that prokaryotic expression obtains can effective renaturation, and then prepare high activity genetic engineering
Horseshoe crab blood G-factor, the present invention go back while establishing corresponding preparation method, are established for the development and application of follow-up fungal infection detection reagent
Basis is determined.
The first purpose of the invention is to provide a kind of horseshoe crab blood G-factor α subunit genes of codon optimization.
Second object of the present invention is to provide a kind of horseshoe crab blood G-factor beta subunit gene of codon optimization.
Third object of the present invention is to provide a kind of recombinant plasmids.
Fourth object of the present invention is a kind of genetic engineering host strain.
Fifth object of the present invention is to provide said gene, above-mentioned recombinant plasmid or said gene engineering host strains to make
Application in standby genetic engineering horseshoe crab blood G-factor.
Sixth object of the present invention is to provide the preparation methods of said gene engineering horseshoe crab blood G-factor.
The present invention the 7th purpose be to provide using the preparation-obtained genetic engineering horseshoe crab blood G of above-mentioned preparation method because
Son.
The 8th purpose of the present invention is to provide said gene engineering horseshoe crab blood G-factor and contains in preparation (1-3)-callose
Measure the application in detection reagent or fungal infection detection reagent.
To achieve the goals above, the present invention is achieved by the following technical programs：
The present invention provides a kind of horseshoe crab blood G-factor α subunit genes of codon optimization, nucleotide sequence such as SEQ ID NO：1
It is shown.
The present invention also provides a kind of horseshoe crab blood G-factor beta subunit gene of codon optimization, nucleotide sequence such as SEQ ID
NO：Shown in 2.
G-factor is a kind of special serine stretch protein proenzyme, is heterodimer, is made of two subunits of α and β.The present invention
By numerous studies, according to the codon preference of Escherichia coli, using the equivalent gene codon phase of Escherichia coli preference
It replaces original horseshoe crab gene codon with answering while keeping the protein sequence expressed constant；The present invention is by by 2 subunits
Gene is cloned again after carrying out codon optimization, the suitable expression vector of reselection and host strain, is successfully realized recombination G-factor and is existed
The solubility expression of high yield in prokaryotes.When the recombination G-factor of gene engineering method production of the present invention is used as detection reagent,
Endotoxic interference is can avoid, false positive is reduced, is especially suitable for early diagnosis fungal infection.In addition, artificial recombination G-factor can be kept away
Exempt from that horseshoe crab resource is consumed excessively and relied on, has sustainable development potentiality.
The present invention also provides a kind of recombinant plasmids, the nucleotides sequence containing above-mentioned α subunit genes and/or beta subunit gene
Row.
Preferably, the carrier of the recombinant plasmid is pET32a-suom.
The present invention also provides a kind of genetic engineering host strain, host strain contains above-mentioned recombinant plasmid.
Preferably, host strain BL21（DE3）.
Correspondingly, α subunit genes and/or beta subunit gene, the above-mentioned recombinant plasmid or above-mentioned engineering bacteria of above-mentioned optimization are being made
Application in standby genetic engineering horseshoe crab blood G-factor, also within protection scope of the present invention.
The present invention also provides a kind of preparation method of genetic engineering horseshoe crab blood G-factor, be by the α subunit genes and/or
The beta subunit gene is separately connected prokaryotic expression carrier, after obtaining recombinant plasmid pETsuom- α and pETsuom- β, then by it
It is converted respectively into the competent cell of Escherichia coli, after expanding culture, IPTG induced expressions is added, take inclusion body, through being denaturalized,
Purifying with merge after, obtain horseshoe crab blood G-factor albumen.
Specifically, the preparation method of the genetic engineering horseshoe crab blood G-factor, includes the following steps：
S1. α subunit gene optimizations are obtained by codon optimization（Nucleotide sequence such as SEQ ID NO：Shown in 1）With the Asias β
Base gene optimization sequence（Nucleotide sequence such as SEQ ID NO：Shown in 2）；
S2. the sequence after above-mentioned codon optimization is connected in pET32a-sumo Vector, it is successful obtains two structures
Recombinant plasmid pETsuom- α and pETsuom- β；
S3. recombinant plasmid pETsuom- α and pETsuom- β are converted respectively to e. coli bl21 (DE3) competent cell
In, obtain engineering bacteria；
S4. culturing engineering bacterium, the IPTG that final concentration of 1 mmol/L is added carry out induced expression；
S5. it is crushed thalline, precipitation is taken, obtains inclusion body；
S6. inclusion body is urea-denatured with 8 M, destination protein is purified using NI-NTA columns after gradient dialysis；
S7. soluble destination protein α subunits and β subunits are obtained with sumo digestions, then by destination protein α subunits and β subunits with
Molar ratio 1：1 fusion obtains compound, as genetic engineering horseshoe crab blood G-factor.
Preferably, in step S4, the temperature of culturing engineering bacterium is 37 DEG C.
It is highly preferred that in step S4, the condition of culturing engineering bacterium is：In 37 DEG C of violent shake cultures of constant-temperature table.
Particularly preferably, in step S4, the method for culturing engineering bacterium is：Picking single bacterium colony adds 5 mL to contain from conversion bacterium colony
The LB culture medium shake cultures of Amp are stayed overnight；Next day takes 5 μ L to be incubated overnight bacterium, and 2 mL LB culture mediums are added（Containing Amp）, it is placed in
37 DEG C of violent shake cultures of constant-temperature table.
Preferably, in step S4, culturing engineering bacterium to A595nm values is up to 0.4~0.6（That is 2~4 h）When, IPTG is added and lures
It leads.
It is highly preferred that in step S4, culturing engineering bacterium to A595nm values is up to 0.5（That is 3 h）When, IPTG inductions are added.
Preferably, in step S4, continue 2~4 h of culture after IPTG inductions are added（It is preferred that 3 h）When, bacterium is collected, is used
SDS-PAGE electrophoresis carries out expression analysis to the foreign protein in precipitation, supernatant and conversion crude extract.
The present invention also provides use the preparation-obtained genetic engineering horseshoe crab blood G-factor of above-mentioned preparation method.
It is verified by experiments, it is poly- that the genetic engineering horseshoe crab blood G-factor that the present invention is prepared can be used for detecting the Portugals (1-3)-β-D-
Sugared content is also used for clinical diagnosis deep fungal infection.And use condition can use at ambient temperature without particular/special requirement.
Therefore, the present invention also provides the genetic engineering horseshoe crab blood G-factors in preparation (1-3)-callose content
Application in detection reagent or fungal infection detection reagent.
Compared with prior art, the present invention has the advantages that：
The present invention by carrying out codon optimization to horseshoe crab blood G-factor α subunit genes and beta subunit gene, be successfully realized horseshoe crab blood G because
The high-yield expression of sub- α subunit genes and beta subunit gene in prokaryotes, to re-assembly out higher concentration with it is active
Horseshoe crab blood G-factor.The present invention uses biological engineering means, and successfully obtaining can be in α, β subunits reconstitution of E. coli
Then two subunits that its induced expression obtains are re-assemblied fusion, by corresponding purification process, obtain purity by plasmid
For 95% or more horseshoe crab blood G-factor albumen；When the horseshoe crab blood G-factor albumen is used as G reagents, endotoxic interference is can avoid, is reduced
False positive is especially suitable for early diagnosis fungal infection, relatively successfully solves G reagent manufactures raw material sources deficiency, testing result
Insecure practical problem is both required equipment and simple for process, quickly, is easy large-scale production, greatly reduces fungi sense
Contaminate the production cost of detection kit.
Description of the drawings
Fig. 1 is pETsuom- alpha expressions in BL21 (DE3) transformed bacteria；Wherein, M is protein standard, and 1 is to turn
Change crude extract（It is induced without IPTG）, 2 be conversion crude extract（IPTG is induced）, 3 be the supernatant after centrifugation（IPTG is induced）, 4 are
Precipitation after centrifugation（IPTG is induced）, 5 be 8 M urea dissolving precipitation（IPTG is induced）.
Fig. 2 is pETsuom- β expression in BL21 (DE3) transformed bacteria；Wherein, M is protein standard, and 1 is to turn
Change crude extract（It is induced without IPTG）, 2 be conversion crude extract（IPTG is induced）, 3 be the supernatant after centrifugation（IPTG is induced）, 4 are
Precipitation after centrifugation（IPTG is induced）, 5 be 8 M urea dissolving precipitation（IPTG is induced）.
Fig. 3 purifies for pETsuom- alpha proteins；Wherein, M be protein standard, 1 be unpurified albumen, 2
For dialyzate, 3 be ni-sepharose purification as a result, 4 be sumo digestion results.
Fig. 4 is the digestion after purification of pETsuom- beta proteins；Wherein, M is protein standard, and 1 is ni-sepharose purification
As a result, 2 flow through liquid for nickel column, 3 wash liquid, 4 label protein to be eluted after digestion, 5 purpose to be eluted after digestion for nickel column
Albumen：The β subunits of G-factor albumen.
Fig. 5 is the genetic engineering horseshoe crab blood G-factor and fluorogenic substrate reaction result figure that the present invention is prepared.
Fig. 6 is the result figure when genetic engineering horseshoe crab blood G-factor of the present invention is detected for fungal infection.
Specific implementation mode
The present invention is made with reference to specific embodiment and further being elaborated, the embodiment is served only for explaining this
Invention, is not intended to limit the scope of the present invention.Test method used in following embodiments is normal unless otherwise specified
Rule method；Used material, reagent etc., unless otherwise specified, for the reagent and material commercially obtained.
The codon optimization of embodiment 1 horseshoe crab blood G-factor α subunit genes and beta subunit gene
The present embodiment is by numerous studies, according to the codon preference of Escherichia coli, using the synonymous base of Escherichia coli preference
Because codon correspondingly replaces original horseshoe crab gene codon while keeping the protein sequence expressed constant.
The nucleotide sequence of α subunit genes after codon optimization such as SEQ ID NO：Shown in 1, the α subunit bases before optimization
The original series of cause such as SEQ ID NO：Shown in 3；
The nucleotide sequence of beta subunit gene after codon optimization such as SEQ ID NO：Shown in 2, beta subunit gene before optimization
Original series such as SEQ ID NO：Shown in 4.
The preparation method of 2 genetic engineering horseshoe crab blood G-factor of embodiment
1, the acquisition of recombinant plasmid
The α subunit genes after codon optimization will be obtained in embodiment 1 and beta subunit gene is building up to pET32a- respectively
In sumo Vector, recombinant plasmid pETsuom- α and pETsuom- β are obtained.
2, the induced expression of recombinant plasmid
Successful recombinant expression plasmid will be built to be transformed into E.coli BL21 (DE3) competence, in the LB containing ampicillin
12 h are cultivated on solid culture plate, single bacterium colony of the picking containing recombinant plasmid adds LB culture medium shake cultures of 5 mL containing Amp to stay overnight,
Next day takes 5 μ L to be incubated overnight bacterium, adds 2mlLB culture mediums（Containing Amp）, it is placed in 37 DEG C of violent shake cultures of constant-temperature table.It waits for
A595nm values are up to 0.5（About 3 hours）When, a pipe plus IPTG to final concentration of 1 mmol/L carry out induced expression, and another pipe is not added with
IPTG is as a contrast.After two pipes continue culture 3 hours, bacterium is collected respectively in 1.5 mL Ep pipes, using SDS-PAGE electricity
It swims and expression analysis is carried out to the foreign protein in precipitation, supernatant and conversion crude extract.
3, the purification renaturation of albumen
4 DEG C of 6500 g centrifuges 15 min and collects above-mentioned expression thalline, and 0.1 times of volume of culture 1 × IB wash buffer is added
And lysozyme（Final concentration of 100 μ g/mL）Fully suspend, be placed at room temperature for 15 min, on ice ultrasonication, 4 DEG C of 1000 g from
10 min of the heart collects precipitation, is resuspended in the 0.1 mol/L Tris-cl that urea concentration is 8M（PH is 8.0）Solution in.With big
It dialyses albumen in the dialyzate of 50 times of sample volume or more, dialyzate 1（200 10 × binding of mL buffer are diluted to 2
Simultaneously 200 μ L 1M final concentration of 0.1 mM of DTT, DTT are added in L, 4M urea, and 8.0,4 DEG C of 4 h or more of dialysis of pH replace dialysis
Liquid 2（200 10 × binding of mL buffer are diluted to 2 L and 200 μ L 1M DTT, DTT final concentration of 0.1 are added
MM, 2M urea, pH 8.0）Dialyse 4 h or more again, to be free of 4 h of DTT and the dialysis of urea dialysis liquid 3.Dialyzate 3 is passed through
Equilibrated NI-NTA columns elute foreign protein with containing 20 mM imidazoles liquid, and destination protein is collected in eluent with 250 mM imidazoles,
With SDS- Polyacrylamide Gel Electrophoresis sample purities.
4, the collection of the cutting of fusion protein and destination protein
Fusion tag is cut on the NI-NTA columns of purifying protein.After dialyzate upper prop, 1 is added by 1000 UL of fusion protein
μ L SUMO enzymes, 4 DEG C overnight.It is added after 1 × IB wash buffer collection digestions and penetrates liquid containing destination protein.Use SDS-
Polyacrylamide Gel Electrophoresis purpose band digestion situation detects albumen concentration with BCA boxes.
3 genetic engineering horseshoe crab blood G-factor biological activity of embodiment is verified
（1）Method
Enzyme fluorogenic substrate method detects enzymatic activity：Boc-Glu- (OBzl)-Gly-Arg-MCA fluorogenic substrates of 5.4 mg are dissolved in
It in 760 μ L DMSO solutions, is kept in dark place in -20 DEG C, a concentration of 10 mM of storing liquid.Respectively to the recombination G-factor of 90 μ L, α
Subunit and β subunits（Albumen concentration is 100 μ g/mL）5 μ L 1mM fluorogenic substrates and 5 μ L 2000 are separately added into sample
Pg/mL (1-3)-callose is uniformly mixed, and after being put into 37 DEG C of 30 min of water-bath, 80 DEG C of 5 min of fire extinguishing are quickly cooled to
Room temperature.The output of product MCA, excitation wavelength lambda ex=380 nm, emission wavelength lambda em=440 nm are measured with microplate reader.
（2）As a result
α the and β subunits of individualism are inactive, cannot be activated by (1-3)-callose.α and β subunits are assembled into G
After factor holoenzyme structure, (1-3)-callose can be special to its after G-factor activation with activated gene engineering horseshoe crab blood G-factor
Substrate is hydrolyzed, this substrate is one section of special polypeptide（Substrate sequence：Boc-Glu-(OBzl)-Gly-Arg-MCA.MCA
It is fluorescent material, fluorescence can be sent out after being hydrolyzed）, polypeptide one fluorescent chemicals of link, the G-factor that polypeptide is activated
Fluorescent material is released after hydrolysis, fluorescent material can be detected that fluorescence is stronger by instrument, and it is more to indicate that G-factor is activated,
Also suggest that (1-3)-callose content in sample is more.
As shown in figure 5, α the and β subunits fluorescence of individualism is or not by force, and to be assembled into G-factor complete for α and β subunits of the present invention
It can be activated by (1-3)-callose after enzymatic structure, a large amount of substrates that hydrolyze release fluorescent material.The experiment results show that this hair
The bright genetic engineering horseshoe crab blood G-factor that high activity has successfully been prepared.
A kind of 4 recombination G-factor tachypleus amebocyte lysate box of embodiment and its detection method
1, a kind of recombination G-factor tachypleus amebocyte lysate box, consisting of：
A bottles：The recombination G-factor and Boc-Glu- (OBzl)-Gly-Arg-MCA fluorogenic substrates being prepared containing embodiment 3（
Chromogenic substrate and diazonium coupling object can be used to substitute）Artificial recombination G-factor reagents；
B bottles：Reagent redissolves liquid；
C bottles：Sample diluting liquid.
D bottles：(1-3)-callose solution.
2, detection method
（1）Acquire fasting subject 1~2 mL of venous blood, by heparin tube with 400 g centrifuge 10 min, take 0.1 mL blood plasma or
Serum dilutes 10 times with apirogen water, and 75 DEG C of 10 min of incubation, taking-up is cooled to room temperature, you can obtains sample test liquid；
（2）By the detection sample of 90 μ L（4 groups of sample point：Healthy People blood sample, monilial infection blood sample（Fungal infections）、
Water and (1-3)-callose solution）Contain the recombination G-factor and Boc-Glu- that embodiment 3 is prepared with 10 μ L
(OBzl) after-Gly-Arg-MCA fluorogenic substrates (A bottles of solution) mixing, 37 DEG C of 30 min of reaction, 80 DEG C inactivate 5 min, fast quickly cooling
But to room temperature；
（3）The output of product MCA, excitation wavelength lambda ex=380 nm, emission wavelength lambda em=440 nm are measured with microplate reader.Note
Meaning：If haemolysis cannot occur in sample.
3, testing result
As shown in fig. 6, Healthy People blood sample fluorescence intensity is substantially less than monilial infection person's blood sample, (1-3)-callose
Solution（Positive control）Group fluorescence intensity enhancing, also showing G-factor can be molten by fungal metabolite (1-3)-callose
Liquid activates, and simple water sample（Negative control object）Fluorescence intensity is very low, these meet expection.This prompt recombination G because
Son can be used for detecting fungal infection in blood sample.
Sequence table
<110>Guangdong medical university
<120>A kind of genetic engineering horseshoe crab blood G-factor and its preparation method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1965
<212> DNA
<213>Horseshoe crab blood G-factor (Limulus Factor G)
<400> 1
tctcacgaac cgaaatggca gctggtttgg tctgacgaat tcaccaacgg tatctcttct 60
gactgggaat tcgaaatggg taacggtctg aacggttggg gtaacaacga actgcagtac 120
taccgtcgtg aaaacgctca ggttgaaggt ggtaaactgg ttatcaccgc taaacgtgaa 180
gactacgacg gtttcaaata cacctctgct cgtctgaaaa cccagttcga caaatcttgg 240
aaatacggta aaatcgaagc taaaatggct atcccgtctt tccgtggtgt ttgggttatg 300
ttctggatgt ctggtgacaa caccaactac gttcgttggc cgtcttctgg tgaaatcgac 360
ttcatcgaac accgtaacac caacaacgaa aaagttcgtg gtactatcca ctggtctacc 420
ccggacggtg ctcacgctca ccacaaccgt gaatctaaca ccaacggtat cgactaccac 480
atctactctg ttgaatggaa ctcttctatc gttaaatggt tcgttaacgg taaccagtac 540
ttcgaagtta aaatccaggg tggtgttaac ggtaaatctg ctttccgtaa caaagttttc 600
gttatcctga acatggctat cggtggtaac tggccgggtt tcgacgttgc tgacgaagct 660
ttcccggcta aaatgtacat cgactacgtt cgtgtttacc aggacgcttc tacctcttct 720
ccggttggtg acacctctct ggacggttac tacttcgttc agaaccgtca ctctgaactg 780
tacctggacg ttaccgacgc ttctaacgaa gacggtgctt tcctgcagca gtggtcttac 840
tctggtaacg aaaaccagca gttcgacttc gaacacctgg aaaacaacgt ttacaaaatc 900
accaacaaaa aatctggtaa atctctggac gtttacaact tcggtactga aaacggtgtt 960
cgtatccagc agtggtctta cggtggtgct cgtaaccagc agttcaccgt tcagtctgtt 1020
ggtgacggtt actacaaaat catcccgcgt ggttctggta aactggttga agttgctgac 1080
ttctctaaag acgctggtgg taaaatccag cagtggtctg acaacaacca gctgtctggt 1140
cagtggaaac tgatcaaatc taaatcttac tctaaactga tccaggctga atcttacttc 1200
gactcttcta aagttcagct ggaagacacc tctgacgttg gtggtggtaa aaacgttaaa 1260
tgcgacaacg aaggtgcttg gatggcttac aaagacatcg acttcccgtc ttctggtaac 1320
taccgtatcg aataccgtgt tgcttctgaa cgtgctggtg gtaaactgtc tctggacctg 1380
aacgctggtt ctatcgttct gggtatgctg gacgttccgt ctaccggtgg ttggcagaaa 1440
tggaccacca tctctcacac cgttaacgtt gactctggta cttacaacct gggtatctac 1500
gttcagcgtg cttcttggaa catcaactgg atcaaaatca ccaaaatccc ggaacagtct 1560
aacctgaacc agggtcgtcg taactctaaa ctgatccagg ctgaatctta cttctcttac 1620
tctgaagttc agctggaaga caccctggac gttggtggtg gtaaaaacgt taaatgcgac 1680
aaagaaggtg cttggatggc ttacaaagac atcgacttcc cgtcttctgg ttcttaccgt 1740
gttgaatacc gtgttgcttc tgaacgtgct ggtggtaaac tgtctctgga cctgaacgct 1800
ggttctatcg ttctgggtat gctggacatc ccgtctaccg gtggtctgca gaaatggacc 1860
accatctctc acatcgttaa cgttgacctg ggtacttaca acctgggtat ctacgttcag 1920
aaagcttctt ggaacatcaa ctggattcgt atcaccaaag tttaa 1965
<210> 2
<211> 837
<212> DNA
<213>Horseshoe crab blood G-factor (Limulus Factor G)
<400> 2
ggtatcaacg aaaaacactg cggtttccgt ccggttatca cccgtatcat cggtggtggt 60
atcgctaccc cgcactcttg gccgtggatg gttggtatct tcaaagttaa cccgcaccgt 120
ttcctgtgcg gtggttctat catcaacaaa gtttctgttg ttaccgctgc tcactgcctg 180
gttacccagt tcggtaaccg tcagaactac tctatcttcg ttcgtgttgg tgctcacgac 240
atcgacaact ctggtactaa ctaccaggtt gacaaagtta tcgttcacca gggttacaaa 300
caccactctc actactacga catcggtctg atcctgctgt ctaaaccggt tgaatacaac 360
gacaaaatcc agccggtttg catcccggaa ttcaacaaac cgcacgttaa cctgaacaac 420
atcaaagttg ttatcaccgg ttggggtgtt accggtaaag ctaccgaaaa acgtaacgtt 480
ctgcgtgaac tggaactgcc ggttgttacc aacgaacagt gcaacaaatc ttaccagacc 540
ctgccgttct ctaaactgaa ccgtggtatc accaacgaca tgatctgcgc tggtttcccg 600
gaaggtggta aagacgcttg ccagggtgac tctggtggtc cgctgatgta ccagaacccg 660
accaccggtc gtgttaaaat cgttggtgtt gtttctttcg gtttcgaatg cgctcgtccg 720
aacttcccgg gtgtttacac ccgtctgtct tcttacgtta actggctgca ggaaatcacc 780
ttcggtcagt ctctggcttc tctgttcgaa gttgttccga tcttcatccc ggaatga 837
<210> 3
<211> 1965
<212> DNA
<213>Horseshoe crab blood G-factor (Limulus Factor G)
<400> 3
agccacgaac caaagtggca gctcgtctgg tcggatgaat ttaccaatgg aataagttct 60
gattgggaat ttgaaatggg caatggcctc aatggttggg gtaataacga actgcaatat 120
tatcgtcgtg aaaatgccca agttgaggga gggaaactgg taattactgc taaaagagaa 180
gactatgatg gcttcaaata cacttctgct aggctgaaaa cccagtttga taaatcttgg 240
aagtatggta aaattgaagc caaaatggcg attccatcat ttcggggagt ctgggtgatg 300
ttctggatgt caggagacaa cactaattat gttagatggc catcttctgg tgaaattgac 360
tttattgaac atagaaacac taacaatgaa aaagtcagag gaactattca ctggtccact 420
cctgacggtg ctcatgcgca tcataacaga gaaagtaata caaatgggat tgattatcac 480
atttattctg tagagtggaa ttcttccatt gttaaatggt ttgttaatgg aaatcaatac 540
tttgaagtga aaattcaggg aggagtaaat gggaaaagtg catttcgtaa caaagttttc 600
gttattttaa acatggcgat tggtggaaac tggccaggat tcgatgttgc tgacgaggct 660
ttccctgcta aaatgtacat tgattatgtc cgtgtatacc aggatgccag tacatcttct 720
cctgttgggg atacctcttt agatggttac tattttgtcc aaaacaggca cagtgaattg 780
tatcttgatg tcactgatgc cagtaacgaa gatggagcat ttctgcaaca atggtcttat 840
agtggtaatg agaaccaaca gtttgatttt gagcatctcg aaaataatgt ttataaaatt 900
actaataaaa aaagtggaaa atctttggat gtttataatt ttgggactga gaatggtgtt 960
agaatccaac agtggtcata tggaggggct cgcaatcagc agtttactgt acaaagtgtt 1020
ggtgatggtt attataagat tattccacgc ggcagtggaa agttagtgga agtagcagat 1080
tttagtaaag atgcaggagg gaagatacaa caatggtctg ataacaacca attatctgga 1140
cagtggaaac ttattaaaag taaaagttat tctaaattaa ttcaggcaga aagttatttt 1200
gattcctcaa aagtacaatt ggaagatacc tcagatgtag gaggtgggaa gaatgttaaa 1260
tgtgataatg aaggagcctg gatggcttat aaggatattg atttccccag ttcaggtaat 1320
tatcgaatag aatacagagt agcaagtgaa cgtgcaggag gaaagctgtc tctggatttg 1380
aatgcaggct ctatagttct tggcatgctg gatgttcctt caacaggagg atggcagaag 1440
tggaccacca tttcccatac agtgaatgtg gattcaggta catataactt ggggatctat 1500
gttcaacgag ccagctggaa tatcaactgg ataaagatta caaaaatacc tgaacagtca 1560
aatttgaatc aagggcgtcg taattctaaa ttaattcagg cagaaagtta ttttagttac 1620
tcagaagtac aactggaaga taccttagat gtaggaggtg gaaagaatgt taaatgtgat 1680
aaagaagggg cctggatggc ttacaaggat attgatttcc ccagttcagg aagttatcga 1740
gtagaataca gagtggcaag tgaacgtgca ggaggaaagc tgtccctaga tttgaatgca 1800
ggctctatag tgcttggcat gctggatatt ccttcaacag gaggattgca gaagtggacc 1860
accatttctc atatagtgaa tgtggattta ggtacatata acttgggaat ttatgttcaa 1920
aaagccagtt ggaatatcaa ttggattaga attacaaaag tgtag 1965
<210> 4
<211> 837
<212> DNA
<213>Horseshoe crab blood G-factor (Limulus Factor G)
<400> 4
ggaataaatg aaaaacattg tgggttccga ccagtaatta caagaattat tggtggagga 60
atagcgacgc ctcattcatg gccgtggatg gttggaattt tcaaagtaaa tcctcaccgt 120
ttcctttgtg gtggatctat tattaataaa gtctctgttg ttactgccgc ccattgtctt 180
gtgacgcagt ttggaaacag acagaattat tctatcttcg taagagttgg agcccatgac 240
atagacaatt cgggtacaaa ttatcaagtg gataaagtta ttgttcacca gggctacaaa 300
caccattcac actactacga tatcggtttg attttactct cgaaaccagt cgaatacaac 360
gacaaaatac agcctgtctg tattcctgag ttcaacaaac ctcacgtgaa cttgaacaat 420
attaaggtcg tcattactgg ttggggtgtt actgggaaag ctactgagaa acgtaacgtt 480
cttcgtgaat tggagttgcc cgtggttaca aacgaacagt gcaacaaatc ttatcagaca 540
ctcccattct caaaattgaa ccgaggaatc actaacgaca tgatttgtgc ggggtttccg 600
gaaggaggga aagatgcttg tcagggcgac tctggtggtc ccctgatgta tcagaatcca 660
acaacaggaa gagtgaaaat agttggagtt gtatcatttg ggttcgaatg tgctcgtccc 720
aacttccccg gtgtttacac gcgcctctcg agctacgtta actggctcca ggaaatcacc 780
ttcggacagt cactcgcttc tttatttgaa gttgtaccaa tatttatacc cgagtga 837
Claims (9)
1. the horseshoe crab blood G-factor α subunit genes and beta subunit gene of a kind of codon optimization, which is characterized in that its nucleotide sequence point
Not such as SEQ ID NO：1 shown and SEQ ID NO：Shown in 2.
2. a kind of recombinant plasmid, which is characterized in that containing described in claim 1 such as SEQ ID NO：1 shown and/or SEQ
ID NO：Nucleotide sequence shown in 2.
3. recombinant plasmid according to claim 2, which is characterized in that its carrier is pET32a-suom.
4. a kind of genetic engineering host strain, which is characterized in that host strain contains the recombinant plasmid described in claim 2.
5. genetic engineering host strain according to claim 4, which is characterized in that host strain BL21（DE3）.
6. genetic engineering host strain exists described in recombinant plasmid or claim 4 described in gene, claim 2 described in claim 1
Prepare the application in genetic engineering horseshoe crab blood G-factor.
7. a kind of preparation method of genetic engineering horseshoe crab blood G-factor, which is characterized in that by α subunit genes and β described in claim 1
Subunit gene is separately connected prokaryotic expression carrier, after obtaining recombinant plasmid pETsuom- α and pETsuom- β, is then distinguished
In conversion to the competent cell of Escherichia coli, after expanding culture, IPTG induced expressions is added, inclusion body are taken, through being denaturalized, purifying
After merging, horseshoe crab blood G-factor albumen is obtained.
8. a kind of preparation-obtained genetic engineering horseshoe crab blood G-factor of preparation method using described in claim 7.
9. genetic engineering horseshoe crab blood G-factor according to any one of claims 8 is in preparation (1-3)-callose content detecting reagent or very
Bacterium infects the application in detection reagent.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116640710A (en) * | 2022-10-08 | 2023-08-25 | 科赫生物科技（北京）有限公司 | Strain for producing horseshoe crab coagulation factor FG beta', preparation method and application |
| CN116640225A (en) * | 2022-10-08 | 2023-08-25 | 科赫生物科技（北京）有限公司 | Limulus blood coagulation factor combination, reaction system, kit and application thereof |
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|---|---|---|---|---|
| WO2008004674A1 (en) * | 2006-07-07 | 2008-01-10 | Seikagaku Corporation | PRO-CLOTTING ENZYME, AND METHOD FOR DETECTION OF ENDOTOXIN OR (1→3)-β-D-GLUCAN USING THE SAME |
| US20100112668A1 (en) * | 2005-03-28 | 2010-05-06 | Seikagaku Corporation | Method For Producing Factor G Derived From HorseShoe Crab |
| CN106119271A (en) * | 2016-07-04 | 2016-11-16 | 长春理工大学 | A kind of DNA recombinant expression Tachypleus tridentatus C factor specific detection endotoxin strengthens turbidimetry |
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2018
- 2018-04-23 CN CN201810369186.6A patent/CN108588084B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100112668A1 (en) * | 2005-03-28 | 2010-05-06 | Seikagaku Corporation | Method For Producing Factor G Derived From HorseShoe Crab |
| WO2008004674A1 (en) * | 2006-07-07 | 2008-01-10 | Seikagaku Corporation | PRO-CLOTTING ENZYME, AND METHOD FOR DETECTION OF ENDOTOXIN OR (1→3)-β-D-GLUCAN USING THE SAME |
| CN106119271A (en) * | 2016-07-04 | 2016-11-16 | 长春理工大学 | A kind of DNA recombinant expression Tachypleus tridentatus C factor specific detection endotoxin strengthens turbidimetry |
Non-Patent Citations (1)
| Title |
|---|
| NORIAKI SEKI ET AL: "Horseshoe crab (1,3)-beta-D-glucan-sensitive coagulation factor G. A serine protease zymogen heterodimer with similarities to beta-glucan-binding proteins", 《THE JOURNAL OF BIOLOGICAL CHAELMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116640710A (en) * | 2022-10-08 | 2023-08-25 | 科赫生物科技（北京）有限公司 | Strain for producing horseshoe crab coagulation factor FG beta', preparation method and application |
| CN116640225A (en) * | 2022-10-08 | 2023-08-25 | 科赫生物科技（北京）有限公司 | Limulus blood coagulation factor combination, reaction system, kit and application thereof |
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| CN108588084B (en) | 2020-11-03 |
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