CN105866080A - Recombinant limulus three-factor reagent and method for detecting endotoxin with same - Google Patents
Recombinant limulus three-factor reagent and method for detecting endotoxin with same Download PDFInfo
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Abstract
The invention discloses a recombinant limulus three-factor reagent which comprises the following components: endotoxin-free water, a heat-resource-free Tris buffer liquid, Tween-20, a fluorescent substrate, a recombinant limulus factor C, a recombinant limulus factor B and a recombinant limulus proclotting enzyme factor. The invention provides the recombinant limulus three-factor reagent and a method for detecting endotoxin with the same, three recombinant limulus factors (the proclotting enzyme factor, the recombinant limulus factor B and the recombinant limulus factor C) can be expressed by using an insect baculovirus expression system, the reagent can be used for detecting bacterial endotoxin, and by adopting the method, bypass interference of a factor G can be eliminated, so that the possibility of false positive caused by glucan is effectively avoided; moreover, the defects that the number of limulus is limited and different batches of limulus reagents are not stable are avoided, and thus the reagent can be used as a novel endotoxin detection reagent.
Description
Technical field
The present invention relates to bioengineering field, particularly relate to a kind of restructuring king crab three factor agents and detect endotoxic
Method.
Background technology
Endotoxin (endotoxin) is the main component of gram negative bacteria (G-) mantle, is also called lipopolysaccharide
(Lipopolysaccharide, LPS).Endotoxin is just released after only dissolving when bacterial death or destroy bacterium cell by artificial means
Releasing, endotoxin is exogenous pyrogen, and it can activate neutrophilic granulocyte etc., is allowed to discharge a kind of endogenous pyrogen,
Acting on thermotaxic centre and cause heating, shock is even dead, thus endotoxin detection is pharmaceutical factory pharmacy and armarium system
Necessary one program in making.
Tachypleus amebocyte lysate (Limulus Amebocyte Lysate, LAL/TachypleusAmebocyte Lysate, TAL) be the world up to
Detect endotoxic goldstandard till the present, there is simple, quick, sensitivity high.It is widely used in detection injection
The bacterial endotoxin that the gram negative bacteria polluted in agent and Implantable Medical Device produces, and the dialysis solution of Clinical practice and
Bacteria endotoxin content in blood.But, tachypleus amebocyte lysate can only produce by collecting king crab hemolymph, along with biological product, doctor
Medicine is such as: preparation and the doctors such as injection medicament, chemical drugs category, radiopharmaceutical, antibiotics, vaccine, dialysis solution
Treat the production units such as apparatus (such as disposable syringe, implantable biomaterial) to increase rapidly, quantitative endotoxin detectable
Demand increasing so that in ocean, the quantity of king crab gradually decreases.
Tachypleus amebocyte lysate main component includes C factor (Factor C, FC), Factor B (Factor B, FB), G-factor, coagulase
Former (Proclotting enzyme), coagulagen (or chromogenic substrate) and bivalent cation, buffer salt etc..The level of its enzyme
Connection reactive group present principles is widely studied, including two agglutination approach: one is the C factor approach of endotaxin mediate:
C factor (FC) combines endotoxin and is activated, and then activates Factor B, and the Factor B of activation is by proclotting enzyme (Proclotting
Enzyme) being converted into coagulase (clotting enzyme), coagulagen is converted into coagulated protein, coagulated protein by coagulase
It is cross-linked with each other and is dehydrated and forms gel；Another is by 1, G-factor (FG) approach of 3-callose mediation, equally
Cause similar agglutination, as shown in Fig. 1 (a), produce false positive results.
In sum, develop not against using king crab hemolymph, be used for detecting endotoxic quick and reliable method and become more
Come the most important.
Tachypleus amebocyte lysate bacterial endotoxins test is divided by reaction principle and can be divided into gel method, nephelometry, chromogenic substrate method at present.Bar
The advantages such as shape virus expression systems has high efficient expression, and safety is good, and expression product is stable, post-treatment modification, with antibacterial,
Yeast, mammalian cell expression system are listed as the big expression system of genetic engineering 4 generally acknowledged.We utilize this system elder brother
Worm cell is expressed king crab proclotting enzyme, B and C factor, and analyzes expression product endotoxin detection activity, it is intended to be low for exploitation
Cost novel endotoxin detectable lays the foundation.
Summary of the invention
It is an object of the invention to provide a kind of restructuring king crab three factor agents and detect endotoxic method, by utilizing insecticide bar
Shape virus expression systems give expression to recombinate king crab three factor (the proclotting enzyme factor, Factor B and C factor), uses three kinds recombinate because of
Son, as its effective active composition, detects endotoxin, the activation by lipopolysaccharide recombinant C factor, the recombinant C factor activator of activation
Restructuring Factor B, proclotting enzyme is converted into coagulase by the Factor B of activation, and fluorogenic substrate is cracked by coagulase, produces glimmering
Recovery compound, as shown in Fig. 1 (b), to quantitative determine fluorescent composition to quantify bacterial endotoxin, the method eliminates G
The factor bypass interference, thus efficiently avoid glucosan produce false-positive probability, and solve king crab limited amount,
The instable shortcoming of each batch of tachypleus amebocyte lysate, can be as novel endotoxin detectable.Compare in restructuring single-factor (C factor)
Toxins checking method, endotoxin detectable of the present invention can produce Cascaded amplification reaction so that endotoxin detection is more efficient
With sensitive.
In order to realize object of the present invention and further advantage, it is provided that a kind of restructuring king crab three factor agents, it comprises following group
Point: without endotoxin water；The buffer of Tris without thermal source；Tween 20；Fluorogenic substrate；Restructuring Limulus factor C；Restructuring king crab B because of
Son；The restructuring king crab proclotting enzyme factor.
Preferably, it comprises the component of following weight portion:
Preferably, it comprises the component of following weight portion:
Preferably, described restructuring Limulus factor C, restructuring king crab Factor B and the restructuring king crab proclotting enzyme factor are by using elder brother
Worm baculovirus expression system is expressed and is obtained.
Preferably, the concentration of described tween 20 is 0.5～1.2 ‰.
Preferably, the concentration of described fluorogenic substrate is 2～4mM.
Preferably, the pH scope of the described buffer of Tris without thermal source is 6.5～7.5.
Preferably, described restructuring Limulus factor C, restructuring king crab Factor B and the restructuring king crab proclotting enzyme factor specifically use such as
Lower method obtains:
Step 1) extract RNA total in limulus blood, by reverse transcription and round pcr to Limulus factor C, king crab Factor B with
And the king crab proclotting enzyme factor expands respectively, respectively obtain the Limulus factor C of PCR amplification, the king crab Factor B of PCR amplification
And the king crab proclotting enzyme factor gene fragment of PCR amplification；
Step 2) provide pTA2 carrier, respectively by PCR expand Limulus factor C, PCR amplification king crab Factor B and
The genetic fragment of the king crab proclotting enzyme factor of PCR amplification is connected on described pTA2 carrier, respectively obtains the first recombiant plasmid；
Described every kind of first recombiant plasmid is sequentially passed through screening, extracts, identify and double digestion, respectively obtain the first product；
Step 3) the first carrier is provided, described every kind of first product obtained is connected respectively on described first carrier,
To the second recombiant plasmid；
Step 4) respectively by described every kind of second recombinant plasmid transformed competence colibacillus cell DH10Bac, and sequentially pass through screening,
Extracting and qualification, respectively obtain bacmid restructuring rod granule；
Step 5) every kind of bacmid will obtaining recombinates rod granule transfection insect cell, uses insect cell medium to cultivate,
To recombinant baculovirus, by the recombinant baculovirus obtained first with the viral infective dose infected insect cell that MOI is 0.1,
By 4 subinfections, respectively obtain restructuring Limulus factor C, restructuring king crab Factor B and the restructuring king crab proclotting enzyme factor；
Wherein, described step 3) in, when described first product is the Limulus factor C obtained by double digestion or king crab proclotting enzyme
Factor gene fragment, described first carrier is pFastBac1；When described first product be the king crab B that obtained by double digestion because of
Mrna exon fragment, described first carrier is pFastBac-Dual；
Described step 5) in, the described insect cell medium of employing is through hyperfiltration treatment.
It is a further object to provide a kind of endotoxic method of detection, comprise the steps:
Step 1) configuration at least 6 variable concentrations endotoxin titer；
Step 2) the configuration endotoxic reagent of detection, and press the volume of 1:1 with each described endotoxin titer respectively
Mixing, obtains mixed liquor, is placed in by mixed liquor in 96 orifice plates；
Step 3) do negative control experiments group, sample controls test group, positive control test group simultaneously；
Step 4) to arrange microplate reader reaction temperature be 37 DEG C, excitation wavelength 360nm, launches wavelength 460nm, response time
It it is 1 hour；
Step 5) to take the logarithm as vertical coordinate with fluorescence intensity change value (RFU), endotoxin concentration of standard solution is taken the logarithm and is marked
Directrix curve.
Preferably, in described step 3) in negative control experiments, its solution is respectively through 50KD's and 10KD
Ultrafiltration post filters.
The present invention at least includes following beneficial effect:
(1) present invention by utilize insect baculovirus expression system build a kind of can in insect cell high efficient expression
King crab factor C, king crab factor B and the restructuring rod granule of king crab factor proclotting enzyme, then with every kind of restructuring rod granule infected insect cell,
Express through amplification and obtain restructuring king crab factor C, king crab factor B and king crab factor proclotting enzyme three factor, use this restructuring three factor
Reagent as endotoxin detection can produce Cascaded amplification reaction when detecting endotoxin so that endotoxin detection is more efficient
With sensitive, can complete detection in 30min, its sensitivity can reach 0.005EU/mL；
(2) restructuring king crab three factor of the present invention can substitute for traditional tachypleus amebocyte lysate, had both solved marine animal king crab quantity drastically
The problem reduced, turn avoid the endotoxin detection false positive that tradition tachypleus amebocyte lysate produces due to G-factor interference, can conduct
A kind of novel endotoxin detectable；
(3) restructuring king crab three factor of the present invention is relative to traditional tachypleus amebocyte lysate, and the composition of endotoxin detectable is single, can
Produce and strict quality control to use biotechnology to carry out, thus it is unstable to avoid the batch caused by raw material
Property, use insect baculovirus expression system to express restructuring king crab three factor simultaneously, only need to gather a small amount of limulus blood cell without
Use king crab hemolymph to produce, protect sea these ancient " living fossil " species of king crab, and can be rapidly with low-cost production
Comprise the endotoxin detectable of these restructuring king crab three factors.
Part is embodied by the further advantage of the present invention, target and feature by description below, and part also will be by the present invention
Research and practice and be understood by the person skilled in the art.
Accompanying drawing explanation
In Fig. 1, (a) is tradition tachypleus amebocyte lysate endotoxin Cleaning Principle figure of the present invention；B () is of the present invention heavy
Group king crab three factor endotoxin Cleaning Principle figure；
In Fig. 2, (a) is the structure schematic diagram of king crab Factor C genes insect expression vector；B () is king crab factor B gene elder brother
The structure schematic diagram of worm expression vector；C () is the structure schematic diagram of king crab factor proclotting enzyme gene insect expression vector；
Sf9 cell schematic diagram in Fig. 3, under (a) microscope；B the Sf9 cell of () transfection king crab factor C restructuring rod granule shows
It is intended to；The Sf9 cell schematic diagram of (c) transfection king crab factor B restructuring rod granule；(d) transfection king crab factor proclotting enzyme restructuring rod granule
Sf9 cell schematic diagram；
In Fig. 4, the solution of (a) negative control experiments group schematic diagram before and after 50KD ultrafiltration post processes；(b)) negative
The solution of controlled trial group schematic diagram before and after 10KD ultrafiltration post processes；(c) endotoxin detection Dynamic Fluorescence curve, bent
Line be corresponding in turn to from top to bottom endotoxin concns be 10,1,0.5,0.1,0.05,0.01,0.005,0EU/ml；D () marks
Directrix curve.
Detailed description of the invention
The present invention is described in further detail below in conjunction with the accompanying drawings, to make those skilled in the art with reference to description word energy
Enough implement according to this.
Should be appreciated that used herein such as " have ", " comprising " and " including " term do not allot one or many
Other element individual or the existence of a combination thereof or interpolation.
Restructuring king crab three factor in the present invention is at the Limulus factor C recombinated respectively, king crab Factor B, the king crab proclotting enzyme factor,
Additionally, Limulus factor C, king crab Factor B, the king crab proclotting enzyme factor hereinafter can be referred to as respectively factor C, factor B,
Factor proclotting enzyme.Factor C, factor B, factor proclotting enzyme can be common be referred to as the factor.
In view of the important meaning of endotoxin detection, and the problem that marine animal king crab quantity drastically reduces, the invention provides one
Planting novel endotoxin detectable restructuring king crab three factor agents, it comprises following component: without endotoxin water；Without thermal source
Tris buffer；Tween 20；Fluorogenic substrate；Restructuring Limulus factor C；Restructuring king crab Factor B；The restructuring king crab proclotting enzyme factor.
Here, the example of king crab includes the one of rounded tail king crab, limulus polyphemus, tachypleus gigas or Tachypleus tridentatus, in these king crabs, and the three of above-mentioned king crab
The preferred Tachypleus tridentatus of the factor.
As a more excellent embodiment of the present invention, described restructuring king crab three factor agents comprises the component of following weight portion:
As a more excellent embodiment of the present invention, described restructuring king crab three factor agents comprises the component of following weight portion:
As a more excellent embodiment of the present invention, described restructuring Limulus factor C, restructuring king crab Factor B and restructuring king crab coagulate
Gu each of proenzyme factor obtains, baculovirus expression by using insect baculovirus expression system to express
System is a kind of eukaryotic system, utilizes the expression vector of polyhedrin strong promoter component in baculovirus structural gene, can
Making a lot of eucaryon genes of interest obtain effectively the most high-caliber expression, the baculovirus used in present expression system does not has spy
Not limiting, mainly this baculovirus with infected insect cell, and thus can express the factor, and can suitably make
With being often used in the baculovirus of expressing protein in insect cell, described baculovirus preferred Autographa californica multicapsid nucleopolyhedrosisvirus caryogram is polygonal
Precursor virus (AcMNPV), described insect cell is not particularly limited, as long as this cell can express the described factor, and
The cell being generally used for expressing heterologous albumen can be suitably used, the example of such insect cell include Sf9, Sf21,
SF+ and Hing-Five, the preferred Sf9 of described insect cell.
As a more excellent embodiment of the present invention, the concentration of described tween 20 is 0.5～1.2 ‰, preferably 1 ‰.
As a more excellent embodiment of the present invention, the concentration of described fluorogenic substrate is 2～4mM.
As a more excellent embodiment of the present invention, the pH scope of the described buffer of Tris without thermal source is 6.5～7.5.
As a more excellent embodiment of the present invention, described restructuring Limulus factor C, restructuring king crab Factor B and restructuring king crab coagulate
Gu the method that the proenzyme factor uses insect baculovirus expression system to carry out expressing is as follows:
Step 1) extract RNA total in limulus blood, by reverse transcription and round pcr to Limulus factor C, king crab Factor B with
And the king crab proclotting enzyme factor expands respectively, respectively obtain the Limulus factor C of PCR amplification, the king crab Factor B of PCR amplification
And the king crab proclotting enzyme factor gene fragment of PCR amplification；
Step 2) provide pTA2 carrier, respectively by PCR expand Limulus factor C, PCR amplification king crab Factor B and
The genetic fragment of the king crab proclotting enzyme factor of PCR amplification is connected on described pTA2 carrier, respectively obtains the first recombiant plasmid；
Described every kind of first recombiant plasmid is sequentially passed through screening, extracts, identify and double digestion, respectively obtain the first product；
Step 3) the first carrier is provided, described every kind of first product obtained is connected respectively on described first carrier,
To the second recombiant plasmid；
Step 4) respectively by described every kind of second recombinant plasmid transformed competence colibacillus cell DH10Bac, and sequentially pass through screening,
Extracting and qualification, respectively obtain bacmid restructuring rod granule；
Step 5) every kind of bacmid will obtaining recombinates rod granule transfection insect cell, uses insect cell medium to cultivate,
To recombinant baculovirus, by the recombinant baculovirus obtained first with the viral infective dose infected insect cell that MOI is 0.1,
By 4 subinfections, respectively obtain restructuring Limulus factor C, restructuring king crab Factor B and the restructuring king crab proclotting enzyme factor；
Wherein, described step 3) in, when described first product is the Limulus factor C obtained by double digestion or king crab proclotting enzyme
Factor gene fragment, described first carrier is pFastBac1；When described first product be the king crab B that obtained by double digestion because of
Mrna exon fragment, described first carrier is pFastBac-Dual；
Described step 5) in, the described insect cell medium of employing is through hyperfiltration treatment；Through hyperfiltration treatment, can go
Except endotoxin present in culture medium, improve the detection sensitivity of reagent；
Described step 2) and described step 3) in screening technique use the screening of blue white macula；Shown step 2) and step 4)
Middle qualification uses PCR to identify order-checking.
Another embodiment of the present invention, is the method using mentioned reagent to carry out endotoxin detection, comprises the steps:
Step 1) configuration at least 6 variable concentrations endotoxin titer, preferably 8；
Step 2) the configuration endotoxic reagent of detection, and press the volume of 1:1 with each described endotoxin titer respectively
Mixing, obtains mixed liquor, is placed in by mixed liquor in 96 orifice plates；Experiment finds, under this ratio, and being correlated with of standard curve
Coefficients R2> 0.98, description standard curve chart is perfect close to straight line, and the endotoxin content thus recorded will be more accurate
Really (R2Obtained by software Origin8 matching)；
Step 3) do negative control experiments group, sample controls test group, positive control test group simultaneously；
Step 4) to arrange microplate reader reaction temperature be 37 DEG C, excitation wavelength 360nm, launches wavelength 460nm, response time
It it is 1 hour；
Step 5) to take the logarithm as vertical coordinate with fluorescence intensity change value (RFU), endotoxin concentration of standard solution is taken the logarithm and is marked
Directrix curve, the decision condition of standard curve effectiveness is to work as coefficient R2>0.98；Coefficient of variation CV < 10%；Negative anti-
Should be worth less than least concentration, standard curve is effective.
In technique scheme, in described step 3) in negative control experiments, its solution respectively through 50KD and
The ultrafiltration post of 10KD filters.
The present invention more specifically will be described by embodiment now.
Embodiment 1 present invention recombinates the expression of king crab three factor
Step 1: the acquisition of king crab three factor gene and amplification；
Extract the total serum IgE in limulus blood cell, and reverse transcription becomes cDNA.According to Limulus factor C in GenBank, king crab B because of
The coding region sequence design primer of son and the king crab proclotting enzyme factor, expands three categories of etiologic factors mrna exon fragment by PCR, respectively obtains
Limulus factor C, Limulus factor C and king crab proclotting enzyme factor genes of interest fragment after amplification.
Step 2: the structure of three factor expression carriers of recombinating and clone；
Will amplification after Limulus factor C, king crab Factor B and king crab proclotting enzyme factor genes of interest fragment respectively with pTA2Vector
Under the conditions of room temperature (15～25 DEG C), all connect 30min, obtain the first recombiant plasmid, then every kind of first recombiant plasmid is divided
Not Zhuan Hua DH5 α competent cell, coating is containing the flat board of ammonia benzyl mycin, 37 DEG C of overnight incubation, blue white macula screening picking
Positive monoclonal bacterium colony, 37 DEG C of fluid medium overnight incubation, extract plasmid, carry out PCR qualification and check order, to through surveying
The recombiant plasmid that sequence is correct, carries out double digestion, respectively obtains the first product；
The first product that the Limulus factor C obtained by double digestion or king crab proclotting enzyme factor gene fragment are obtained with
PFastBac-Dual carrier connects, by king crab Factor B genetic fragment the first product obtained by double digestion and pFastBac1
Carrier connects；Connecting temperature is all 16 DEG C, and the Connection Time is 24 hours, respectively obtains the second recombiant plasmid, by every kind
Second recombinant plasmid transformed DH5 α competent cell, coating, containing ammonia benzyl mycin and the flat board of gentamycin, was cultivated for 37 DEG C
Night, blue white macula screening picking positive monoclonal bacterium colony, 37 DEG C of fluid medium overnight incubation, extract plasmid, carry out double enzyme
Cut qualification and check order；
By described every kind of second recombinant plasmid transformed DH10Bac competent cell, coating is containing gentamycin, sulphuric acid card sodium
Mycin and the flat board of tetracycline, 37 DEG C of overnight incubation, blue white macula screening picking positive monoclonal bacterium colony, 37 DEG C of liquid cultures
Base overnight incubation, extracts plasmid, carries out PCR qualification and check order, and respectively obtains bacmid restructuring rod granule.
Step 3: the expression of restructuring king crab three categories of etiologic factors daughter cell；
As it is shown on figure 3, by every kind of Bacmid restructuring rod granule transfection Sf9 cell monolayer (six orifice plates), cell occurs the most sick
During poison infection symptoms, collecting culture supernatant, centrifugal speed 1450r/min, time 5min, the supernatant obtained is P1, takes
P1 takes culture supernatant by MOI value 0.1 infection Sf9 cell and obtains P2, then infects Sf9 cell, then takes culture supernatant, and two
Obtaining the P3 virus of high titre after secondary, plaque assay surveys virus titer；Infect Sf9 cell by P3 generation virus, receive after 72h
Collection culture supernatant, respectively obtains the king crab recombinant C factor, king crab restructuring Factor B and the king crab restructuring proclotting enzyme factor；Consider
Present in cell culture medium, endotoxin may affect the endotoxin detection sensitivity of the restructuring king crab factor, and we will test used
Culture medium carries out hyperfiltration treatment, improves the detection sensitivity of reagent.
Step 4: endotoxic detection；
According to bacterial endotoxin standard substance description, endotoxin is diluted to 0,0.005,0.01,0.05,0.1,0.5,1,
10EU/mL；
Preparation endotoxin reaction reagent: without endotoxin water 40 μ L, without thermal source Tris buffer (pH6.8) 30 μ L, tween 20
(1 ‰ concentration) 10 μ L, fluorogenic substrate (3mM) 10 μ L, the recombinant C factor 10 μ L, recombinate Factor B 10 μ L, and restructuring is solidifying
Gu the proenzyme factor 10 μ L；And press the volume mixture of 1:1 with each described endotoxin titer respectively, obtain mixed liquor,
Mixed liquor is placed in 96 orifice plates；Every hole adds 100 μ L endotoxin reaction reagents and 100 μ L endotoxin standards are (each dense
Spend 3 multiple holes), be negative control experiments group 100 μ L simultaneously, wherein, the solution of negative control group respectively through 50KD with
And the ultrafiltration post of 10KD filters, such as Fig. 4 (a) (the multiple hole of each concentration 3) and Fig. 4 (b) (the multiple hole of each concentration 2)
Shown in, sample controls test group 100 μ L (the multiple hole of each concentration 2), positive control test group 100 μ L (each concentration 2
Individual multiple hole)；
Arranging microplate reader reaction temperature is 37 DEG C, excitation wavelength 360nm, launches wavelength 460nm, and the response time is 1 little
Time, endotoxin detection Dynamic Fluorescence curve, as shown in Fig. 4 (c)；
Taking the logarithm as vertical coordinate with fluorescence intensity change value (RFU), endotoxin normal concentration is taken the logarithm and is done standard curve such as figure
Shown in 4 (d), the decision condition of standard curve effectiveness is to work as coefficient R2>0.98；Coefficient of variation CV < 10%；Cloudy
Property response value less than least concentration, standard curve is effective.
Embodiment 2
The expression of restructuring king crab three factor is identical with embodiment 1, and difference is that in endotoxin detection, endotoxin reacts
The composition of reagent is different, and the constituent content of endotoxin reagent is: without endotoxin water 35 μ L, without thermal source Tris buffer
(pH6.5) 25 μ L, tween 20 (0.5 ‰ concentration) 5 μ L, fluorogenic substrate (2mM) 8 μ L, the recombinant C factor 5 μ L, restructuring B because of
Son 6 μ L, recombinate the proclotting enzyme factor 6 μ L, and other are the most identical.
Embodiment 3
The expression of restructuring king crab three factor is identical with embodiment 1, and difference is that in endotoxin detection, endotoxin reacts
The composition of reagent is different, and the constituent content of endotoxin reagent is: without endotoxin water 40 μ L, without thermal source Tris buffer (pH7.5)
30 μ L, tween 20 (1.2 ‰ concentration) 10 μ L, fluorogenic substrate (4mM) 10 μ L, the recombinant C factor 10 μ L, Factor B of recombinating
10 μ L, recombinate the proclotting enzyme factor 10 μ L, and other are the most identical.
Although embodiment of the present invention are disclosed as above, but it is not restricted in description and embodiment listed fortune
With, it can be applied to various applicable the field of the invention completely, for those skilled in the art, and can be easily
Realizing other amendment, therefore under the general concept limited without departing substantially from claim and equivalency range, the present invention does not limit
In specific details with shown here as the legend with description.
Claims (10)
1. restructuring king crab three factor agents, it is characterised in that it comprises following component:
Water without endotoxin；The buffer of Tris without thermal source；Tween 20；Fluorogenic substrate；Restructuring Limulus factor C；Restructuring king crab B because of
Son；The restructuring king crab proclotting enzyme factor.
2. restructuring king crab three factor agents as claimed in claim 1, it is characterised in that it comprises the component of following weight portion:
3. restructuring king crab three factor agents stated such as claim 1, it is characterised in that it comprises the component of following weight portion:
4. restructuring king crab three factor agents as described in any one in claims 1 to 3, it is characterised in that described restructuring
Limulus factor C, restructuring king crab Factor B and the restructuring king crab proclotting enzyme factor are by using insect baculovirus expression system to carry out
Express and obtain.
5. restructuring king crab three factor agents as claimed in claim 4, it is characterised in that the concentration of described tween 20 is
0.5～1.2 ‰.
6. restructuring king crab three factor agents as claimed in claim 4, it is characterised in that the concentration of described fluorogenic substrate is
2～4mM.
7. restructuring king crab three factor agents as claimed in claim 4, it is characterised in that the described buffer of Tris without thermal source
PH scope is 6.5～7.5.
8. restructuring king crab three factor agents as claimed in claim 4, it is characterised in that described restructuring Limulus factor C, restructuring
King crab Factor B and the restructuring king crab proclotting enzyme factor are specifically adopted and are obtained with the following method:
Step 1) extract RNA total in limulus blood, by reverse transcription and round pcr to Limulus factor C, king crab Factor B with
And the king crab proclotting enzyme factor expands respectively, respectively obtain the Limulus factor C of PCR amplification, the king crab Factor B of PCR amplification
And the king crab proclotting enzyme factor gene fragment of PCR amplification；
Step 2) provide pTA2 carrier, respectively by PCR expand Limulus factor C, PCR amplification king crab Factor B and
The genetic fragment of the king crab proclotting enzyme factor of PCR amplification is connected on described pTA2 carrier, respectively obtains the first recombiant plasmid；
Described every kind of first recombiant plasmid is sequentially passed through screening, extracts, identify and double digestion, respectively obtain the first product；
Step 3) the first carrier is provided, described every kind of first product obtained is connected respectively on described first carrier,
To the second recombiant plasmid；
Step 4) respectively by described every kind of second recombinant plasmid transformed competence colibacillus cell DH10Bac, and sequentially pass through screening,
Extracting and qualification, respectively obtain bacmid restructuring rod granule；
Step 5) every kind of bacmid will obtaining recombinates rod granule transfection insect cell, uses insect cell medium to cultivate,
To recombinant baculovirus, by the recombinant baculovirus obtained first with the viral infective dose infected insect cell that MOI is 0.1,
By 4 subinfections, respectively obtain restructuring Limulus factor C, restructuring king crab Factor B and the restructuring king crab proclotting enzyme factor；
Wherein, described step 3) in, when described first product is the Limulus factor C obtained by double digestion or king crab proclotting enzyme
Factor gene fragment, described first carrier is pFastBac1；When described first product be the king crab B that obtained by double digestion because of
Mrna exon fragment, described first carrier is pFastBac-Dual；
Described step 5) in, the described insect cell medium of employing is through hyperfiltration treatment.
9. the reagent applied as described in any one in claim 4～8 detects endotoxic method, it is characterised in that
Comprise the steps:
Step 1) configuration at least 6 variable concentrations endotoxin titer；
Step 2) the configuration endotoxic reagent of detection, and press the volume of 1:1 with each described endotoxin titer respectively
Mixing, obtains mixed liquor, is placed in by mixed liquor in 96 orifice plates；
Step 3) do negative control experiments group, sample controls test group, positive control test group simultaneously；
Step 4) to arrange microplate reader reaction temperature be 37 DEG C, excitation wavelength 360nm, launches wavelength 460nm, response time
It it is 1 hour；
Step 5) to take the logarithm as vertical coordinate with fluorescence intensity change value (RFU), endotoxin concentration of standard solution is taken the logarithm and is marked
Directrix curve.
10. as claimed in claim 9 detect endotoxic method, it is characterised in that in described step 3) in negative right
According in test, its solution filters respectively through the ultrafiltration post of 50KD and 10KD.
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| CN112462015A (en) * | 2020-11-18 | 2021-03-09 | 海南倍特药业有限公司 | Method for detecting Remazolam hydrobromide bacterial endotoxin |
| CN112462015B (en) * | 2020-11-18 | 2022-07-12 | 海南倍特药业有限公司 | Method for detecting bacterial endotoxin of remazolam hydrobromide |
| 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 |
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Effective date of registration: 20221028 Address after: No. 131, Xinjia Road, Haicang Xinyang Industrial Zone, Xiamen, Fujian 361000 Patentee after: XIAMEN BIOENDO TECHNOLOGY Co.,Ltd. Address before: 215163 Suzhou City, Jiangsu, science and Technology City, No. 88 Patentee before: Suzhou Institute of Biomedical Engineering and Technology Chinese Academy of Sciences |