CN102807972B - Hepatitis A virus purification method - Google Patents
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Abstract
The invention provides a hepatitis A virus purification method. With hepatitis A virus cell culture fluid serving as raw materials, the hepatitis A virus purification technology is completed via the technological processes of ultrasonication, centrifugal removal of cell debris, chloroform extraction, ultra-filtering concentration, hydrophobic chromatography, detection, collection and the like. Inactivated hepatitis A vaccines prepared from the hepatitis A virus cell culture fluid are high in purity and valence, simple in technology, easy to magnify and safe and reliable to use.
Description
Technical field
The present invention relates to the method for purifying virus field, particularly, relate to a kind of method of purification of hepatitis A virus.
Background technology
Hepatitis A, be called for short hepatitis A, is the acute infectious disease of a kind of serious harm human health of being caused by hepatitis A virus (HAV).Hepatitis A is mainly propagated by " excrement-mouth " approach, or the propagation in the individual human world, thereby or causes that hepatitis A breaks out because of water or the food that has polluted hepatitis A virus (HAV).After big-age-child and adult infect hepatitis A, more than 70%, be clinical type infection, case fatality rate is 0.3%～0.6%; More than 50 years old, patient's case fatality rate is 1.8%; After the Chronic Liver patient infects hepatitis A, the danger that acute hepatic failure occurs raises.Along with the improvement of living condition, the grownup infects the hepatitis A number the trend of increasing, and clinical type hepatitis A ratio rises, thereby hepatitis A becomes more serious public health problem.
China is the hepatitis A district occurred frequently, has at least every year 240000 people to suffer from hepatitis A, causes huge financial loss and social danger, and therefore, vaccine and the medicine of developing and develop effectively prevention and treatment hepatitis A become problem demanding prompt solution.
The vaccine of two kinds of prevention and control hepatitis A is arranged at present in the world, and a kind of is Attenuated HAV，Live; Another kind is hav inactivated vaccine.Due to the hepatitis A virus (HAV) purifying process difference that each manufacturer is used, quality product is uneven.
Common hepatitis A virus (HAV) separation method is the sieve chromatography technology at present, this technical characterstic be operate relatively simple, and production application for a long time, but this technology is not high to the purity of hepatitis A virus (HAV) purifying, the hav antigen purity made is lower than 50%, makes that in every dose of hepatitis A inactivated vaccine finished product, total protein concentration is higher than 10 μ g/ml, and this has increased the unsafe factor of Hepatitis A Vaccine, increase the risk that vaccine is used, and increased cost.Therefore in the urgent need to a kind of good to the hepatitis A virus (HAV) purification effect, viral purity is higher, and unit volume chromatography glue purification efficiency is high, and the hepatitis A virus method of purification is beneficial to extensive virus production purifying with the supply production of vaccine safely and effectively.
Summary of the invention
The object of the present invention is to provide a kind of method of purification of hepatitis A virus, to overcome above-mentioned deficiency.
The hepatitis A virus SH strain that the present invention uses, its deposit number is CGMCC No.4501, has been disclosed in Chinese patent CN102174477A.
The invention provides a kind of method of purification of hepatitis A virus, comprise the following steps:
1) hepatitis A virus cell culture fluid ultrasonication, centrifugal removal cell debris, collect supernatant liquor;
2) chloroform extracting supernatant liquor, obtain rough virus liquid;
3) by rough virus liquid ultrafiltration and concentration, buffer salt solution wash-out ultra-filtration membrane;
4) hydrophobic chromatography purifying concentrating virus liquid;
5) detection of virus liquid and collection.
Wherein, the ultrasonication of the described hepatitis A virus cell culture fluid of step 1) is antigen titre to be reached to hepatitis A virus cell culture fluid ultrasonication under condition of ice bath of 1:256-1024.
Wherein, the Ultrasonic Cell Disruptor power that the described ultrasonication of step 1) is used is 500-1500w, and output rating is 50%-95%, and broken number of times is 4-6 time.
Wherein, the centrifugal method of described step 1) is 3500-4500r/min, centrifugal 5min-10min.
Wherein, described step 2) the chloroform extracting be by chloroform by volume for 1:1-3 adds the supernatant liquor jolting, then the centrifugal rear absorption of 3500-4500r/min upper strata water, so repeat extracting 4-6 time; Albumen phase and chloroform, again with the extraction buffer extracting and draw the upper strata water, are merged to the upper strata water and obtain rough virus liquid.
Wherein, described extraction buffer is 120mM NaCl or 6.2mM PBS.
Wherein, the multiple of described step 3) ultrafiltration and concentration is 10-30 times, and the molecular weight cut-off of ultra-filtration membrane is 50-100KD.
Wherein, the PBS solution that described step 3) buffer salt solution is 2-4mol/L, pH7.2.
Wherein, described step 4) chromatography method is: each applied sample amount is 1000-2000ml, and sample-loading buffer is 2mol/L PBS, pH7.2, and elution buffer is 0.5mol/L PBS, pH7.2, flow velocity is 15ml/min.
Further, with the 280nm wavelength on the post good to balance the wash-out virus liquid detected.
The invention provides the application of above-mentioned hepatitis A virus method of purification in preparing Hepatitis A Vaccine.
Will by the hydrophobic chromatography collection step to virus liquid carry out deactivation, according to volume ratio 1:4000-5000, formaldehyde is added in virus liquid, 37 ℃ of constant temperature, rotate and within 12-15 days, complete inactivation process.
The deactivation hepatitis A virus is according to tiring rare joining by after the inspections such as aseptic, effect, undue toxicity, bacterial endotoxin, being the Hepatitis A virus vaccine goods.
The present invention has following useful effect: (1) has improved the hepatitis A virus rate of recovery, and high density buffering salt wash-out ultra-filtration membrane, effectively reduce ultra-filtration membrane to viral absorption; (2) improved viral purity, the cytopathy venom can effectively be removed foreign protein through ultrasonication, chloroform extracting, ultra-filtration membrane ultrafiltration and concentration and gel permeation chromatography, improve viral purity, the purity of the hepatitis A virus that uses the inventive method purifying to obtain is greater than 95%; (3) hepatitis A virus that the inventive method purifying obtains is for the preparation of hepatitis A or vaccine, in the hepatitis A inactivated vaccine finished product of every dose of effective dose, total protein concentration is lower than 3 μ g/ml, more safe and reliable higher than 10 μ g/ml than total protein concentration in prior art.Therefore the virus that the inventive method is purified has safe and reliable for the preparation of Hepatitis A virus vaccine, immunogenicity is good, high specificity.
The accompanying drawing explanation
The VP1 that Fig. 1 is the hepatitis A virus that in embodiment 1, purifying obtains, VP2 and VP3 content detection HPLC color atlas.
The VP1 that Fig. 2 is the hepatitis A virus that in embodiment 2, purifying obtains, VP2 and VP3 content detection HPLC color atlas.
The VP1 that Fig. 3 is the hepatitis A virus that in embodiment 3, purifying obtains, VP2 and VP3 content detection HPLC color atlas.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
The method of purification of embodiment 1 hepatitis A virus and the application (1) for preparing vaccine
1, get 1000ml hepatitis A virus harvest liquid (hepatitis A virus (HAV) SH strain preserving number: CGMCC No.4501).By hepatitis A virus harvest liquid (aseptic antigen titre reaches 1:512) under condition of ice bath, use ultrasonic grinding instrument (purchased from Ningbo Xin Zhi company, rated output 1500w, output rating 1200w) smudge cells temperature, cell crashing ratio is reached more than 99%, ultrasonication 4 times;
2, by the enchylema after ultrasonication with the centrifugal 10min of the rotating speed of 3500r/min, collect supernatant liquor;
3, chloroform by volume 1:3 add jolting in supernatant liquor, the centrifugal absorption of 3500r/min upper strata water; Albumen phase and chloroform are used extraction buffer (6.2mM PBS) extracting again, and adding chloroform, to jolt rear absorption supernatant be 1 extracting, repeat extracting 5 times, merge the upper strata water and obtain rough virus liquid;
4, rough hepatitis A venom step 3 made is through the ultra-filtration membrane ultrafiltration and concentration, 10 times of ultrafiltration and concentration, and the molecular weight cut-off of ultra-filtration membrane is 50KD;
5, use the PBS solution of high density buffer salt solution 2mol/L, pH7.2 wash-out ultra-filtration membrane;
6, concentrated virus liquid is through gel chromatography, and gel chromatography chromatography column used is Phenyl Sepharose 6FF.Chromatography method is: each applied sample amount is 1000ml, and sample-loading buffer is 2mol/LPBS, pH7.2, and elution buffer is 0.5mol/L PBS, pH7.2, flow velocity is 15ml/min.
7, detect and collect virus.With the 280nm wavelength, the virus liquid of XK50/30 post (purchased from GE company) the upper wash-out good to balance is detected (the observation electricity is led), lead observed result according to electricity, lead 30 from electricity and start to collect virus liquid, collect electricity always and lead and drop to 2.4, merge the virus liquid of collecting and complete the purifying technique process.
8, HPLC purity check
Purifying gained HAV liquid in step 7 is added in the mixing solutions that final concentration is 4mol/L hydrochloric acid arc (GuHCL) and lmmol/L dithiothreitol (DTT) (DTT).50 ℃ of water bath with thermostatic control l0min cracking HAV particles.The HPLC chromatographic column is TSK-GEL G5000PW, and moving phase is 6.2mmol/L pH6.8PBS, and flow velocity is 0.6ml/min, the UV-detector wavelength is 280nm, temperature is 25 ℃, and each sample size is 50 μ l, goes out behind peak VP1, VP2 and VP3 protein peak from the raised collection hepatitis A virus of half-peak.By the high performance liquid chromatograph system software analysis, find, VP1, VP2 and VP3 purity reach 97%, wherein VP1 purity 7%, VP2 purity 64%, VP3 purity 26%.The HPLC color atlas is shown in Fig. 1.
9, deactivation.The hepatitis A virus liquid of collecting in step 7 is added to formaldehyde by 1/4000 of its cumulative volume, then put in 37 ℃ of thermostatic chambers, rotate and within 12 days, complete inactivation process.
10, calibrating.The deactivation hepatitis A virus is according to tiring rare joining by after the inspections such as aseptic, effect, undue toxicity, bacterial endotoxin, being the Hepatitis A virus vaccine goods.
(1) vaccine preparation:
After the deactivation of purifying hepatitis A venom, HAV-Ag is tired as 6400EU/ml, uses 2mM PBS(pH7.2) dilute 10 times and be the hepatitis A inactivated vaccine prepared to the HAV-Ag 640EU/ml that tires.
(2) sterility test
In pressing, " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII A Sterility Test carries out.By vaccine with after 0.22 μ m membrane filtration, with 0.1% peptone solution cleaning filter membranes, filter membrane is divided into three parts, puts into respectively 2 sulphur glycollate culture mediums and 1 improvement Martin substratum, wherein 1 sulphur glycollate culture medium is put 30 ℃, and all the other are put 20 ℃ and cultivate 14 days.The sterility test result is qualified.
(3) effect inspection
By " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix X S method, undertaken.Vaccine and HAV-Ag reference material are diluted to identical a series of extent of dilution, use ELISA reagent, measure each dilution absorbancy of vaccine and reference material, according to the parallel lines statistical technique, calculate the relative effectivenes of vaccine with respect to reference material.The vaccine potency that the present embodiment makes is 1.12.
(4) abnormal toxicity tests
By " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII F method, undertaken.The abdominal injection vaccine, injected in mice 0.5ml, totally 5, cavy injection 5.0ml, totally 2.Observe 7 days.Detecting the undue toxicity of finding the present embodiment vaccine is 0.50.
(5) bacterial endotoxin inspection
By " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII E gel test method(s) of limiting the quantity of, undertaken.With tachypleus amebocyte lysate bacterial detection intracellular toxin, vaccine should dilute 80 times, to eliminate the impact of interfering factors.Found that bacterial endotoxin that the present embodiment makes hepatitis A vaccine is for<2.0EU/ml.
The method of purification of embodiment 2 hepatitis A virus and the application (2) for preparing vaccine
1, get 1000ml hepatitis A virus harvest liquid (hepatitis A virus (HAV) virus SH strain preserving number: CGMCC NO.4501).By hepatitis A virus harvest liquid (aseptic antigen titre reaches 1:256) under condition of ice bath, use ultrasonic grinding instrument (purchased from Ningbo Xin Zhi company, rated output 1500w, output rating 1200w) smudge cells temperature, cell crashing ratio is reached more than 99%, ultrasonication 5 times;
2, the enchylema after ultrasonication, with the centrifugal 8min of the rotating speed of 4000r/min, is collected supernatant liquor;
3, chloroform by volume 1:2 add jolting in supernatant liquor, the centrifugal absorption of 4000r/min upper strata water; Albumen phase and chloroform are used extraction buffer (120mM NaCl) extracting again, and adding chloroform, to jolt rear absorption supernatant be 1 extracting, repeat extracting 6 times, merge the upper strata water and obtain rough virus liquid;
4, rough hepatitis A venom step 3 made is through the ultra-filtration membrane ultrafiltration and concentration, 20 times of ultrafiltration and concentration, and the molecular weight cut-off of ultra-filtration membrane is 50KD;
5, use the PBS solution of high density buffer salt solution 3mol/L, pH7.2 wash-out ultra-filtration membrane;
6, concentrated virus liquid is through gel chromatography, and gel chromatography chromatography column used is Phenyl Sepharose 6FF.Chromatography method is: each applied sample amount is 1500ml, and sample-loading buffer is 2mol/LPBS, pH7.2, and elution buffer is 0.5mol/L PBS, pH7.2, flow velocity is 15ml/min.
7, detect and collect virus.With the 280nm wavelength, the virus liquid of XK50/30 post (purchased from GE company) the upper wash-out good to balance is detected (the observation electricity is led), lead observed result according to electricity, lead 30 from electricity and start to collect virus liquid, collect electricity always and lead and drop to 2.4, merge the virus liquid of collecting and complete the purifying technique process.
8, HPLC purity check
Purifying gained HAV liquid in step 7 is added in the mixing solutions that final concentration is 4mol/L hydrochloric acid arc (GuHCL) and lmmol/L dithiothreitol (DTT) (DTT).50 ℃ of water bath with thermostatic control l0min cracking HAV particles.The HPLC chromatographic column is TSK-GEL G5000PW, and moving phase is 6.2mmol/L pH6.8PBS, and flow velocity is 0.6ml/min, the UV-detector wavelength is 280nm, temperature is 25 ℃, and each sample size is 50 μ l, goes out behind peak VP1, VP2 and VP3 protein peak from the raised collection hepatitis A virus of half-peak.By the high performance liquid chromatograph system software analysis, find, VP1, VP2 and VP3 purity reach 98%, wherein VP1 purity 7%, VP2 purity 65%, VP3 purity 26%.The HPLC color atlas is shown in Fig. 2.
9, deactivation.The hepatitis A virus liquid of collecting in step 7 is added to formaldehyde by 1/4500 of its cumulative volume, then put in 37 ℃ of thermostatic chambers, the ROT13 sky completes inactivation process.
10, calibrating.The deactivation hepatitis A virus is according to tiring rare joining by after the inspections such as aseptic, effect, undue toxicity, bacterial endotoxin, being the Hepatitis A virus vaccine goods.
(1) vaccine preparation:
After the hepatitis A venom deactivation of purifying, HAV-Ag is tired as 12800EU/ml, uses 2mM PBS(pH7.2) dilute 20 times and be the hepatitis A inactivated vaccine prepared to the HAV-Ag 640EU/ml that tires.
(2) sterility test
In pressing, " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII A Sterility Test carries out.By vaccine with after 0.22 μ m membrane filtration, with 0.1% peptone solution cleaning filter membranes, filter membrane is divided into three parts, puts into respectively 2 sulphur glycollate culture mediums and 1 improvement Martin substratum, wherein 1 sulphur glycollate culture medium is put 32 ℃, and all the other are put 23 ℃ and cultivate 14 days.The sterility test result that the present embodiment makes vaccine is qualified.
(3) effect inspection
By " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix X S method, undertaken.Vaccine and HAV-Ag reference material are diluted to identical a series of extent of dilution, use ELISA reagent, measure each dilution absorbancy of vaccine and reference material, according to the parallel lines statistical technique, calculate the relative effectivenes of vaccine with respect to reference material.The effect that the present embodiment makes vaccine is 1.05.
(4) abnormal toxicity tests
By " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII F method, undertaken.The abdominal injection vaccine, injected in mice 0.5ml, totally 5, cavy injection 5.0ml, totally 2.Observe 7 days.The undue toxicity that the present embodiment makes vaccine is 0.50.
(5) bacterial endotoxin inspection
By " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII E gel test method(s) of limiting the quantity of, undertaken.With tachypleus amebocyte lysate bacterial detection intracellular toxin, vaccine should dilute 80 times, to eliminate the impact of interfering factors.The bacterial endotoxin that the present embodiment makes vaccine is<2.0EU/ml.
The method of purification of embodiment 3 hepatitis A virus and the application (3) for preparing vaccine
1, get 1000ml hepatitis A virus harvest liquid (hepatitis A virus (HAV) strain preserving number: CGMCC NO.4501).By hepatitis A virus harvest liquid (aseptic antigen titre reaches 1:1024) under condition of ice bath, use ultrasonic grinding instrument (purchased from Ningbo Xin Zhi company, rated output 1500w, output rating 1200w) smudge cells temperature, cell crashing ratio is reached more than 99%, ultrasonication 5 times;
2, the enchylema after ultrasonication, with the centrifugal 5min of the rotating speed of 4500r/min, is collected supernatant liquor;
3, chloroform by volume 1:1 add jolting in supernatant liquor, the centrifugal absorption of 4500r/min upper strata water; Albumen phase and chloroform are used extraction buffer (120mM NaCl) extracting again, and adding chloroform, to jolt rear absorption supernatant be 1 extracting, repeat extracting 4 times, merge the upper strata water and obtain rough virus liquid;
4, rough hepatitis A venom step 3 made is through the ultra-filtration membrane ultrafiltration and concentration, 30 times of ultrafiltration and concentration, and the molecular weight cut-off of ultra-filtration membrane is 100KD;
5, use the PBS solution of high density buffer salt solution 4mol/L, pH7.2 wash-out ultra-filtration membrane;
6, concentrated virus liquid is through gel chromatography, and gel chromatography chromatography column used is Phenyl Sepharose 6FF.Chromatography method is: each applied sample amount is 2000ml, and sample-loading buffer is 2mol/LPBS, pH7.2, and elution buffer is 0.5mol/L PBS, pH7.2, flow velocity is 15ml/min.
7, detect and collect virus.With the 280nm wavelength, the virus liquid of XK50/30 post (purchased from GE company) the upper wash-out good to balance is detected (the observation electricity is led), lead observed result according to electricity, lead 30 from electricity and start to collect virus liquid, collect electricity always and lead and drop to 2.4, merge the virus liquid of collecting and complete the purifying technique process.
8, HPLC purity check
Purifying gained HAV liquid in step 7 is added in the mixing solutions that final concentration is 4mol/L hydrochloric acid arc (GuHCL) and lmmol/L dithiothreitol (DTT) (DTT).50 ℃ of water bath with thermostatic control l0min cracking HAV particles.The HPLC chromatographic column is TSK-GEL G5000PW, and moving phase is 6.2mmol/L pH6.8PBS, and flow velocity is 0.6ml/min, the UV-detector wavelength is 280nm, temperature is 25 ℃, and each sample size is 50 μ l, goes out behind peak VP1, VP2 and VP3 protein peak from the raised collection hepatitis A virus of half-peak.By the high performance liquid chromatograph system software analysis, find, VP1, VP2 and VP3 purity reach 97%, wherein VP1 purity 7%, VP2 purity 64%, VP3 purity 26%.The HPLC color atlas is shown in Fig. 3.
9, deactivation.The hepatitis A virus liquid of collecting in step 7 is added to formaldehyde by 1/5000 of its cumulative volume, then put in 37 ℃ of thermostatic chambers, rotate and within 15 days, complete inactivation process.
10, calibrating.The deactivation hepatitis A virus is according to tiring rare joining by after the inspections such as aseptic, effect, undue toxicity, bacterial endotoxin, being the Hepatitis A virus vaccine goods.
(1) vaccine preparation:
After the hepatitis A venom deactivation of purifying, HAV-Ag is tired as 12800EU/ml, uses 2mM PBS(pH7.2) dilute 20 times and be the hepatitis A inactivated vaccine prepared to the HAV-Ag 640EU/ml that tires.
(2) sterility test
In pressing, " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII A Sterility Test carries out.By vaccine with after 0.22 μ m membrane filtration, with 0.1% peptone solution cleaning filter membranes, filter membrane is divided into three parts, puts into respectively 2 sulphur glycollate culture mediums and 1 improvement Martin substratum, wherein 1 sulphur glycollate culture medium is put 35 ℃, and all the other are put 25 ℃ and cultivate 14 days.The sterility test result that the present embodiment makes vaccine is qualified.
(3) effect inspection
By " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix X S method, undertaken.Vaccine and HAV-Ag reference material are diluted to identical a series of extent of dilution, use ELISA reagent, measure each dilution absorbancy of vaccine and reference material, according to the parallel lines statistical technique, calculate the relative effectivenes of vaccine with respect to reference material.The effect that the present embodiment makes vaccine is 1.10.
(4) abnormal toxicity tests
By " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII F method, undertaken.The abdominal injection vaccine, injected in mice 0.5ml, totally 5, cavy injection 5.0ml, totally 2.Observe 7 days.The undue toxicity that the present embodiment makes vaccine is 0.50.
(5) bacterial endotoxin inspection
By " Chinese people republic pharmacopeia " 2010 editions the 3rd appendix XII E gel test method(s) of limiting the quantity of, undertaken.With tachypleus amebocyte lysate bacterial detection intracellular toxin, vaccine should dilute 80 times, to eliminate the impact of interfering factors.The bacterial endotoxin that the present embodiment makes vaccine is<2.0EU/ml.
Claims (3)
1. the method for purification of a hepatitis A virus comprises the following steps:
1) hepatitis A virus cell culture fluid ultrasonication, centrifugal removal cell debris, collect supernatant liquor, and described centrifugal condition is 3500r/min, centrifugal 10min; Wherein, described ultrasonication is antigen titre to be reached to hepatitis A virus cell culture fluid ultrasonication under condition of ice bath of 1:512, the Ultrasonic Cell Disruptor power rating power used is 1500w, output rating is 1200w, broken number of times is 4 times, cell crashing ratio is reached more than 99%, and described hepatitis A virus is hepatitis A virus (HAV) SH strain, and preserving number is CGMCC No.4501;
2) chloroform extracting supernatant liquor, obtain rough virus liquid; Described chloroform extracting be by chloroform by volume for 1:3 adds the supernatant liquor jolting, then the centrifugal rear absorption of 3500r/min upper strata water, so repeat extracting 5 times; Albumen phase and chloroform, again with the extraction buffer extracting and draw the upper strata water, are merged to the upper strata water and obtain rough virus liquid; Described extraction buffer is 6.2mM PBS;
3) by rough virus liquid ultrafiltration and concentration, buffer salt solution wash-out ultra-filtration membrane, the multiple of ultrafiltration and concentration is 10 times, the molecular weight cut-off of ultra-filtration membrane is 50KD; The PBS solution that buffer salt solution is 2mol/L, pH7.2;
4) hydrophobic chromatography purifying concentrating virus liquid; Described chromatography is: each applied sample amount is 1000ml, and sample-loading buffer is 2mol/L PBS, pH7.2, and elution buffer is 0.5mol/L PBS, pH7.2, flow velocity is 15ml/min;
5) with the 280nm wavelength on the post of the XK50/30 purchased from GE company good to balance the virus liquid of wash-out detected, the observation electricity is led, and according to electricity, leads observed result, leads 30 from electricity and starts to collect virus liquid, collect electricity always and lead and drop to 2.4, merge the virus liquid of collecting and complete the purifying technique process;
The hepatitis A virus liquid of collecting is added to formaldehyde by 1/4000 of its cumulative volume, then put in 37 ℃ of thermostatic chambers, rotate and within 12 days, complete inactivation process; After the deactivation of purifying hepatitis A venom, HAV-Ag is tired as 6400EU/ml, with 10 times of 2mM PBS, pH7.2, dilutions to the HAV-Ag 640EU/ml that tires, after aseptic, effect, undue toxicity, bacterial endotoxin inspection, be the Hepatitis A virus vaccine goods.
2. the method for purification of a hepatitis A virus comprises the following steps:
1) hepatitis A virus cell culture fluid ultrasonication, centrifugal removal cell debris, collect supernatant liquor, and described centrifugal condition is 4000r/min, centrifugal 8min; Wherein, described ultrasonication is antigen titre to be reached to hepatitis A virus cell culture fluid ultrasonication under condition of ice bath of 1:256, the Ultrasonic Cell Disruptor power rating power used is 1500w, output rating is 1200w, broken number of times is 5 times, cell crashing ratio is reached more than 99%, and described hepatitis A virus is hepatitis A virus (HAV) SH strain, and preserving number is CGMCC No.4501;
2) chloroform extracting supernatant liquor, obtain rough virus liquid; Described chloroform extracting be by chloroform by volume for 1:2 adds the supernatant liquor jolting, then the centrifugal rear absorption of 4000r/min upper strata water, so repeat extracting 6 times; Albumen phase and chloroform, again with the extraction buffer extracting and draw the upper strata water, are merged to the upper strata water and obtain rough virus liquid; Described extraction buffer is 120mM NaCl;
3) by rough virus liquid ultrafiltration and concentration, buffer salt solution wash-out ultra-filtration membrane, the multiple of ultrafiltration and concentration is 20 times, the molecular weight cut-off of ultra-filtration membrane is 50KD; The PBS solution that buffer salt solution is 3mol/L, pH7.2;
4) hydrophobic chromatography purifying concentrating virus liquid; Described chromatography is: each applied sample amount is 1500ml, and sample-loading buffer is 2mol/L PBS, pH7.2, and elution buffer is 0.5mol/L PBS, pH7.2, flow velocity is 15ml/min;
5) with the 280nm wavelength on the post of the XK50/30 purchased from GE company good to balance the virus liquid of wash-out detected, the observation electricity is led, and according to electricity, leads observed result, leads 30 from electricity and starts to collect virus liquid, collect electricity always and lead and drop to 2.4, merge the virus liquid of collecting and complete the purifying technique process;
The hepatitis A virus liquid of collecting is added to formaldehyde by 1/4500 of its cumulative volume, then put in 37 ℃ of thermostatic chambers, the ROT13 sky completes inactivation process; After the deactivation of purifying hepatitis A venom, HAV-Ag is tired as 12800EU/ml, with 20 times of 2mM PBS, pH7.2, dilutions to the HAV-Ag 640EU/ml that tires, after aseptic, effect, undue toxicity, bacterial endotoxin inspection, be the Hepatitis A virus vaccine goods.
3. the application of the described method of claim 1 or 2 in preparing Hepatitis A Vaccine.
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