CN101539555B - Method for establishing snake poison fingerprint maps and fingerprint maps thereof - Google Patents
Method for establishing snake poison fingerprint maps and fingerprint maps thereof Download PDFInfo
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Abstract
The invention provides a method for establishing snake poison fingerprint maps, which is characterized by preparing snake poison freeze-dry powder into an aqueous solution with certain concentration, separating and detecting the aqueous solution by a capillary electrophoresis apparatus under certain condition to obtain the fingerprint maps of various components in the snake poison. The invention also provides a standard map obtained by the method, and the standard map can provide a reliable basis for identifying the snake poison and controlling the inherent quality.
Description
Technical field
The present invention relates to a kind of method for building up of snake poison fingerprint maps, and by the standard diagram of this method gained.
Background technology
Snake venom is one type of material from various poisonous snake snakehead poison gland, secreting, as poisonous snake protection oneself, hunts the weapon of food, is one of the most malicious in the world zootoxin.The principal ingredient of snake venom is protein and polypeptide; Account for the 90-95% of dry weight; Comprising plurality of enzymes, like phosphate, arginine ester hydrolysing enzyme, proteolytic enzyme, phospholipase A2, type kallikrein, 5-nucleotidase, L-amino acid oxidase and acetylcholinesterase etc.The non-zymoprotein matter of 3-12 kind or polypeptide are like neurotoxin, film activity peptide, BPP, homorrhagic factor etc.Much have pharmacological action and medical value in these activated compositions, be used for the control of thrombotic disease like the batroxobin in the snake venom, fibrinolysin clinically as medicine for treating thrombus.In addition, also contain some small-molecular peptides, amino acid, carbohydrates, lipid, nucleosides, alkamines and metallic ion etc. in the snake venom.The composition of snake venom has tangible difference because of differences such as the kind of snake, the place of production, time, snake age, male and female, living environments.
Snake venom is that the mankind study and utilize maximum zootoxin up to now, but its early stage research is very arduous, mainly is that separation, the purification ratio because of protein is difficult.After the seventies,,, just be able to the chemical nature of each component of snake venom is studied, and entering is master's a molecular level with structure and function by means of advanced person's biotechnology means along with molecular biology by leaps and bounds develops.Fast development along with biotechnology; In addition to the attention of natural materials; Research to snake venom effective constituent is quite deep; The work of one-component and molecule biological property research having begun to carry out primary structure and gene expression, very definite curative effect has been used for many years and obtained to single component formulations clinically.
The whole world nearly 13 sections of existing snake class, 400 belong to, kind more than 2700, and wherein 1st/4th, poisonous snake.China's snake class resource is very abundant, nearly 8 sections, 53 genus, 219 kinds (comprising subspecies).Kind surplus the poisonous snake about 50 wherein, but severe toxicity is arranged and endanger bigger only kind surplus in the of 10, that is: banked krait, Bungarus multicinctus, cobra, king cobra, circle spot adder, grassland adder, pallas pit viper, agkistrodon acutus, green bamboo snake, solder horn, mountain solder horn.The research of the snake venom of China is started late, and approximately is at the beginning of the eighties, domestic priority in Guangxi, Fujian, Kunming, Anhui etc. have set up the research department, from the snake venom of more than 20 kind of poisonous snakes such as Pallas pit viper section, glasses section, Hydrophiidae, separate, purifying multiple composition.Domesticly now also carry out research to snake venom component molecules structure and function aspects, but research scale and the degree of depth with abroad compare, also have a certain distance.
Principal ingredient in the snake venom is an albumen, and polypeptide reaches bioactivators such as enzyme, the protein that its essence is made up of amino acid.Domestic research for snake venom at present concentrates on wherein some concrete active component mostly, such as Defibrase, and fibrinolysin, and a series of neurotoxicity factors or the like.And do not appear in the newspapers as yet to the finger-print research that snake venom integral body is carried out.Wei generation show etc. utilizes the isotachophoresis technology that cobra venom, cobra-venom and agkistrodon acutus venom are analyzed discriminating since the nineties, and snake poison lyophilized powder and fresh snake venom are compared research; Also snake venom is mixed pseudo-article and carried out identification and analysis, the result shows that the snake venom in different snake kinds, the different places of production or snake venom mix the puppet article, and is different because of its component, content, shows inconsistent on the isotachophoresis collection of illustrative plates; And several indifferences of the electrophoresis pattern of the freeze-dried powder of snake venom of the same race and fresh venom.This research shows that using the isotachophoresis technology carries out discriminatory analysis to snake venom, will play an important role to the quality monitoring of snake venom.Though this method is a kind of effective analytical approach, because the restriction of aspects such as instrument, reagent is used not general in China.
Cao Jinhong etc. study the discriminating of white-browed cobra-venom, black eyebrow abdomen snake venom, agkistrodon acutus venom, trimeresurus stejnegeri snake venom and solder horn snake venom with crossed immunoelectrophoresis.Because the total component kind of various snake venom is different and the difference of each component relative content, precipitation line position and peak relative height ratio etc. have nothing in common with each other in the electrophoresis pattern, can be used for distinguishing different snake venom.But this method need be prepared the antiserum of corresponding snake venom in advance, has suitable limitation.The public Ao in storehouse waits and utilizes two-dimensional immunoelectrophoresis, in conjunction with corresponding snake venom antiserum, also accomplishes certain snake venom evaluation work.
But,, can not adapt to the demand of current snake venom research because above electrophoresis method self repeatability is not good low with resolution.New analysis means is needed badly and is applied to research.
Fingerprint pattern technology comes from fingerprint identification.Being usually used in traditional Chinese medicine research, is a kind of comprehensive, macroscopic view and quantifiable discriminating means, in order to Chinese crude drug and Chinese patent drug are discerned the false from the genuine the homogeneity and the stability of evaluation raw medicinal material, semi-manufacture and end product quality.Its base attribute is " globality " and " ambiguity "." globality " is meant intactly the relatively characteristic " looks " of chromatogram; What " ambiguity " was emphasical is the similarity of reference substance and testing sample finger-print.
Capillary Electrophoresis has with the appearance amount and lacks as a kind of day by day ripe separate mode, environmental friendliness, and theoretical pedal number is high, the resolution advantages of higher.Chang Keyu high performance liquid chromatography (HPLC) complementation is as the research means of finger-print.The object of present domestic utilization capillary electrophoresis separation is confined to micromolecule mostly, and the mode of trials such as the Zhang Xuan of Tsing-Hua University utilization microemulsion kapillary electrokinetic chromatography was carried out preliminary the discussion to the separation condition of protein, but Shang Weijian promotes the use.
In order to seek the effective means of check and analysis snake venom; The thought that the present invention intends with traditional Chinese medicine fingerprint is guidance; Related art method with capillary electrophoresis separation protein is the basis, sets up the finger-print of snake venom, for the discriminating of snake venom and the control of inherent quality provide more reliable foundation.And in the further research snake venom other not principal component reference is provided.
Summary of the invention
The objective of the invention is provides a kind of snake poison fingerprint maps method for building up to the deficiency that has the snake venom detection method now; After being characterized in snake poison lyophilized powder is mixed with the certain density WS; Pass through the separation detection of HPCE under certain conditions, obtain the finger-print of various compositions in the snake venom.Thereby can reliable foundation be provided for the discriminating of snake venom and the control of inherent quality.
Snake poison fingerprint maps method for building up of the present invention specifically comprises the following steps:
1) preparation of need testing solution: get snake poison lyophilized powder, be dissolved in distilled water after, mixing, centrifugal, get supernatant as need testing solution;
2) preparation of reference substance solution: get the fibrinolysin reference substance, be dissolved in distilled water as reference substance solution;
3) make snake poison fingerprint maps: accurate respectively reference substance solution and the need testing solution drawn; Through the HPCE separation detection; Consisting of of electrophoretic buffer wherein: normal octane 0.6%～1.0%; Normal butyl alcohol 4.5%～8.0%, SDS 2.5%～5.0% and borax soln 8～12mmol/L; Demarcate its fingerprint characteristic with relative migration time and relative peak area mean value then, thereby obtain the snake venom standard finger-print.
Wherein, sample solution prepares according to following steps: precision takes by weighing snake venom 15mg, is dissolved in distilled water 1ml, and behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets sample solution.Existing with join at present.
Reference substance solution prepares as follows: precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
Deposition condition is: non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
The composition of electrophoretic buffer is preferably: normal octane 0.64%, normal butyl alcohol 4.62%, SDS3.5% and borax soln 10mmol/L.Jiangsu and Zhejiang Provinces viper venom separating effect is best under this composition condition.
The foundation of Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint (CEFP, Capillary ElectrophoresisFinger Print):
1 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 7666, and separates well with adjacent peak, so be elected to be the object of reference peak.
2 Jiangsu and Zhejiang Provinces viper venom standard C EFP measure
The Jiangsu and Zhejiang Provinces viper venom of 11 different batches is dissolved in distilled water solution.On HPCE, analyze 3 times the record electrophoretogram.Demarcate its fingerprint characteristic with relative migration time and relative peak area mean value.Through to its CEFP comparative studies, be 15 (see figure 1)s by the definite total fingerprint peaks of the total rate f=100% at electrophoresis peak, f=m
i/ n * 100%, m
iBe the sample lot number that the i fingerprint peaks occurs, n is total lot number.
The standard diagram that the present invention also provides the method for building up by above-mentioned snake poison fingerprint maps to obtain.
The advantage of the inventive method is that method is easy, stable, precision is high, favorable reproducibility, be easy to grasp, and can hold the kind and the quality situation of snake venom from the global feature looks of chromatogram, can be with it as one of index of snake venom quality control and real and fake discrimination.
Description of drawings
Fig. 1 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under embodiment 1 condition;
Fig. 2 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under embodiment 2 conditions;
Fig. 3 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under embodiment 3 conditions;
Fig. 4 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under embodiment 4 conditions;
Fig. 5 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under embodiment 5 conditions;
Fig. 6 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under embodiment 6 conditions;
Fig. 7 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under embodiment 7 conditions;
Fig. 8 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under embodiment 8 conditions;
Fig. 9 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under embodiment 9 conditions;
Figure 10 is the agkistrodon halys ussuriensis snake venom capillary electrophoresis fingerprint that obtains under embodiment 10 conditions;
Figure 11 is the long-noded pit viper snake venom capillary electrophoresis fingerprint that obtains under embodiment 11 conditions;
Figure 12 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under Comparative Examples 1 condition;
Figure 13 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under Comparative Examples 2 conditions;
Figure 14 is the Jiangsu and Zhejiang Provinces viper venom capillary electrophoresis fingerprint that obtains under Comparative Examples 3 conditions.
Embodiment
Embodiment 1
1 need testing solution and reference substance solution preparation
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.64%, normal butyl alcohol 4.62%, SDS 3.5% and borax soln 10mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 7666, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated Jiangsu and Zhejiang Provinces viper venom CEFP (seeing table 1) with transit time, relative migration time, peak area, relative peak area mean value, Jiangsu and Zhejiang Provinces viper venom standard C EFP sees Fig. 1.
Table 1
Embodiment 2
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.6%, normal butyl alcohol 6.5%, SDS 3.5% and borax soln 10mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 7308, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated Jiangsu and Zhejiang Provinces viper venom CEFP (seeing table 2) with transit time, relative migration time, peak area, relative peak area mean value, Jiangsu and Zhejiang Provinces viper venom standard C EFP sees Fig. 2.
Table 2
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kY; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 1.0%, normal butyl alcohol 6.5%, SDS 3.5% and borax soln 10mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 6927, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated Jiangsu and Zhejiang Provinces viper venom CEFP (seeing table 3) with transit time, relative migration time, peak area, relative peak area mean value, Jiangsu and Zhejiang Provinces viper venom standard C EFP sees Fig. 3.
Table 3
Embodiment 4
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.8%, normal butyl alcohol 4.5%, SDS 3.5% and borax soln 10mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 7395, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated Jiangsu and Zhejiang Provinces viper venom CEFP (seeing table 4) with transit time, relative migration time, peak area, relative peak area mean value, Jiangsu and Zhejiang Provinces viper venom standard C EFP sees Fig. 4.
Table 4
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.8%, normal butyl alcohol 8.0%, SDS 3.5% and borax soln 10mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 8142, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated Jiangsu and Zhejiang Provinces viper venom CEFP (seeing table 5) with transit time, relative migration time, peak area, relative peak area mean value, Jiangsu and Zhejiang Provinces viper venom standard C EFP sees Fig. 5.
Table 5
Embodiment 6
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.8%, normal butyl alcohol 6.5%, SDS 2.5% and borax soln 10mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 6260, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated Jiangsu and Zhejiang Provinces viper venom CEFP (seeing table 6) with transit time, relative migration time, peak area, relative peak area mean value, Jiangsu and Zhejiang Provinces viper venom standard C EFP sees Fig. 6.
Table 6
Embodiment 7
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kY; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.8%, normal butyl alcohol 6.5%, SDS 5.0% and borax soln 10mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 7243, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated Jiangsu and Zhejiang Provinces viper venom CEFP (seeing table 7) with transit time, relative migration time, peak area, relative peak area mean value, Jiangsu and Zhejiang Provinces viper venom standard C EFP sees Fig. 7.
Table 7
Embodiment 8
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.8%, normal butyl alcohol 6.5%, SDS 3.5% and borax soln 8mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 6982, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated Jiangsu and Zhejiang Provinces viper venom CEFP (seeing table 8) with transit time, relative migration time, peak area, relative peak area mean value, Jiangsu and Zhejiang Provinces viper venom standard C EFP sees Fig. 8.
Table 8
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.8%, normal butyl alcohol 6.5%, SDS 3.5% and borax soln 12mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 7172, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated Jiangsu and Zhejiang Provinces viper venom CEFP (seeing table 9) with transit time, relative migration time, peak area, relative peak area mean value, Jiangsu and Zhejiang Provinces viper venom standard C EFP sees Fig. 9.
Table 9
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of agkistrodon halys ussuriensis snake venom, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.64%, normal butyl alcohol 4.62%, SDS 3.5% and borax soln 10mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and agkistrodon halys ussuriensis snake venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 8 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the fibrinolysin peak is 8729, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 agkistrodon halys ussuriensis snake venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated agkistrodon halys ussuriensis snake venom CEFP (seeing table 10) with transit time, relative migration time, peak area, relative peak area mean value, agkistrodon halys ussuriensis snake venom standard C EFP sees Figure 10.
Table 10
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of long-noded pit viper snake venom, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing Defibrase reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.64%, normal butyl alcohol 4.62%, SDS 3.5% and borax soln 10mmol/L.
4 system suitability test and object of reference qualitative
With Defibrase reference substance solution and long-noded pit viper snake venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 9 peaks are Defibrase.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.The theoretical pedal number that records the Defibrase peak is 8497, and separates well with adjacent peak, so be elected to be the object of reference peak.
5 long-noded pit viper snake venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, demarcated long-noded pit viper snake venom CEFP (seeing table 11) with transit time, relative migration time, peak area, relative peak area mean value, long-noded pit viper snake venom standard C EFP sees Figure 11.
Table 11
Comparative Examples 1
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 0.45%, normal butyl alcohol 3.5%, SDS 1.5% and borax soln 6mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 13 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.So be elected to be the object of reference peak.But the theoretical pedal number that records the fibrinolysin peak is merely 2577, separates not good with adjacent peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, measured Jiangsu and Zhejiang Provinces viper venom CEFP (seeing accompanying drawing 12).It is thus clear that each peak separation degree is scarcely good, can't accurately measure transit time, relative migration time, peak area, the relative peak area at each peak by figure.So this condition should not be used to formulate snake venom standard C EFP.
Comparative Examples 2
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 40cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 1.2%, normal butyl alcohol 8.5%, SDS 5.5% and borax soln 15mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 16 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.So be elected to be the object of reference peak.But the theoretical pedal number that records the fibrinolysin peak is merely 1569, separates not good with adjacent peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, measured Jiangsu and Zhejiang Provinces viper venom CEFP (seeing accompanying drawing 13).It is thus clear that each peak separation degree is scarcely good, can't accurately measure transit time, relative migration time, peak area, the relative peak area at each peak by figure.So this condition should not be used to formulate snake venom standard C EFP.
Comparative Examples 3
The preparation of 1 need testing solution
Get each 15mg mixing of 11 batches of Jiangsu and Zhejiang Provinces viper venoms, be dissolved among the distilled water 10ml, behind the ultrasonic concussion mixing, (10000rpm 10min), gets supernatant to high speed centrifugation, promptly gets need testing solution.Existing with join at present.
Precision takes by weighing fibrinolysin reference substance 10mg, is dissolved in distilled water 1ml as reference substance solution;
2 deposition conditions
Non-coatings capillary pipe 75 μ m * 35cm, effective length 25cm (Hebei light transmitting fiber Yongnian factory); Ultraviolet detection wavelength 254nm; Working voltage 15kV; 25 ℃ of capillary temperatures; Input mode 0.5psi, 5s.Use methyl alcohol successively before using new capillary column, 1mol/L HCl, 1mol/L NaOH, each 10min of purified rinse water capillary column.Use 0.1mol/L NaOH and purified rinse water capillary column 10min respectively successively before experiment beginning every day with after finishing.Use 0.1mol/L NaOH before the each run sample successively, pure water, running buffer, flushing 7min under 20psi.
3 electrophoretic buffers are selected
Damping fluid is formed: normal octane 1.2%, normal butyl alcohol 8.5%, SDS 1.5% and borax soln 6mmol/L.
4 system suitability test and object of reference qualitative
With fibrinolysin reference substance solution and Jiangsu and Zhejiang Provinces viper venom test liquid at HPCE upward pressure sample introduction 5s.The contrast transit time can know that No. 11 peaks are fibrinolysin.In snake venom sample test liquid, add reference substance solution and analyze, electrophoresis peak gain results proves that above-mentioned conclusion is correct.So be elected to be the object of reference peak.But the theoretical pedal number that records the fibrinolysin peak is merely 2774, separates not good with adjacent peak.
5 Jiangsu and Zhejiang Provinces viper venom standard C EFP make
The need testing solution sample introduction is analyzed 3 times, measured Jiangsu and Zhejiang Provinces viper venom CEFP (seeing accompanying drawing 14).It is thus clear that each peak separation degree is scarcely good, can't accurately measure transit time, relative migration time, peak area, the relative peak area at each peak by figure.So this condition should not be used to formulate snake venom standard C EFP.
Claims (7)
1. the method for building up of snake poison fingerprint maps after it is characterized in that snake poison lyophilized powder is mixed with the WS, through the separation detection of HPCE, obtains the finger-print of various compositions in the snake venom;
Said method comprises the steps:
1) preparation of need testing solution: get snake poison lyophilized powder, be dissolved in distilled water after, mixing, centrifugal, get supernatant as need testing solution;
2) preparation of reference substance solution: get the fibrinolysin reference substance, be dissolved in distilled water as reference substance solution;
3) make snake poison fingerprint maps: accurate respectively reference substance solution and the need testing solution drawn; Through the HPCE separation detection; Consisting of of electrophoretic buffer wherein: normal octane 0.6%～1.0%; Normal butyl alcohol 4.5%～8.0%, SDS 2.5%～5.0% and borax soln 8～12mmol/L; Demarcate its fingerprint characteristic with relative migration time and relative peak area mean value then, thereby obtain the snake venom standard finger-print.
2. the method for building up of snake poison fingerprint maps as claimed in claim 1 is characterized in that the condition of said HPCE separation detection is: ultraviolet detection wavelength 195nm; Working voltage 10kV; 18 ℃ of capillary temperatures.
3. the method for building up of snake poison fingerprint maps as claimed in claim 1; It is characterized in that said need testing solution prepares as follows: precision takes by weighing each 15mg of various snake venom, is dissolved in distilled water 1ml, behind the ultrasonic concussion mixing; Speed high speed centrifugation 10min with 10000rpm gets supernatant.
4. the method for building up of snake poison fingerprint maps as claimed in claim 1, it is characterized in that said reference substance solution prepares as follows: precision takes by weighing fibrinolysin reference substance 10mg, is dissolved among the distilled water 1ml.
5. the method for building up of snake poison fingerprint maps as claimed in claim 1 is characterized in that each sample introduction 5s of said need testing solution and reference substance solution.
6. the method for building up of snake poison fingerprint maps as claimed in claim 1 is characterized in that consisting of of said electrophoretic buffer: normal octane 0.64%, normal butyl alcohol 4.62%, SDS 3.5% and borax soln 10mmol/L.
7. the finger-print that is obtained by the method for building up of each described snake poison fingerprint maps of claim 1 to 6 after it is characterized in that snake poison lyophilized powder is mixed with the WS, through the separation detection of HPCE, obtains the finger-print of various compositions in the snake venom.
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