CN112813121B

CN112813121B - Method for preparing tuberculin pure protein derivative by using mycobacterium tuberculosis low virulent strain H37Ra

Info

Publication number: CN112813121B
Application number: CN202110144419.4A
Authority: CN
Inventors: 孙超伟
Original assignee: Chengdu Keen Biotechnology Co ltd
Current assignee: Chengdu Keen Biotechnology Co ltd
Priority date: 2021-02-02
Filing date: 2021-02-02
Publication date: 2022-05-10
Anticipated expiration: 2041-02-02
Also published as: CN112813121A

Abstract

The invention discloses a method for preparing a tuberculin pure protein derivative by using a mycobacterium tuberculosis low virulent strain H37Ra, belonging to the technical field of biological protein preparation. The tuberculin pure protein derivative is prepared by taking a mycobacterium tuberculosis low-virulent strain CMCC93020(H37Ra) as a strain, and performing amplification, inactivation, salting-out precipitation, sterilization filtration, inspection, dilution and split charging. The method overcomes the defects of great harm of strains to operators and environment, high requirements on production conditions and the like in the prior art (P3 production workshop), and finally obtains the protein which can be used for clinical diagnosis of tuberculosis, screening of BCG vaccination objects, monitoring of organism immune reaction after BCG vaccination and epidemiological monitoring.

Description

Method for preparing tuberculin pure protein derivative by using mycobacterium tuberculosis low virulent strain H37Ra

Technical Field

The invention relates to a method for preparing a tuberculin pure protein derivative by using a mycobacterium tuberculosis low virulent strain H37Ra, belonging to the technical field of biological protein preparation.

Background

Tuberculosis is a chronic infectious disease that seriously harms human and animal health, in which mycobacterium tuberculosis is the causative bacterium of tuberculosis. 140 thousands of tuberculosis deaths worldwide in 2019, and tuberculosis still serves as a killer of the first infectious disease worldwide. According to 2020 annual report of tuberculosis worldwide released by WTO: the number of the tuberculosis in the world is estimated to be about 1000 ten thousand in 2019 (890-; among them, 264 million in India accounts for 26%; indonesia 84.6 ten thousand, 8.5%; 83.3 thousands of China, accounting for 8.4 percent. China is the third world rank of tuberculosis people, and is also a tuberculosis high-burden country.

The number of patients with pulmonary tuberculosis in China is large, and according to the result of fifth tuberculosis epidemiological sampling survey in China in 2010, the method has the following advantages: the prevalence rate of active tuberculosis of people over 15 years old in China is 459/10 ten thousand, the prevalence rate of masculine tuberculosis is 66/10 ten thousand, and the prevalence rate of bacterial masculine tuberculosis is 119/10 ten thousand. Accordingly, the number of active tuberculosis, daubing yang and junyang patients currently suffered by the population over 15 years old in the country is estimated to be 499 thousands, 72 thousands and 129 thousands respectively; the prevalence rates of the rural pulmonary tuberculosis are all obviously higher than that of cities and towns, and the prevalence rates of the active pulmonary tuberculosis, the inunction pulmonary tuberculosis and the bacterial-yang pulmonary tuberculosis in the western regions are all obviously higher than that in the middle and the eastern regions. According to official statistics, the carriers of tubercle bacillus in China are about 5.5 hundred million, and active non-tuberculous patients are about 500 ten thousand.

Therefore, the previous screening and diagnosis of tuberculosis becomes one of the most important in tuberculosis prevention. At present, the commercially available tuberculin diagnostic reagent is mainly tuberculin pure protein derivative (TB-PPD) produced by beijing auspicious biological products limited, and the company discloses a patent document with a publication number of CN109182167A entitled "a high-potency tuberculin skin test diagnostic reagent (PPD) production process" in 2019, 11.01 month, wherein the patent documents specifically disclose: comprises the steps of bacteria culture, inactivation and precipitation; the specific process of the bacterial culture comprises the following steps: firstly, the working seed strain is cultured and developed into a light yellow lawn which is dry-wrinkled and conglobated, the microscopic examination state of the lawn is rough bacillus brevis, and the two ends of the lawn are slightly bent; then 2 generations of culture are carried out to the membrane with the most wrinkles and the yellowish fungus membranes, 3 generations of culture are carried out when the surface of the culture medium is fully paved with the fungus membranes and the culture solution is clear and transparent, and 4 generations of culture are carried out for 8-10 weeks after 3 generations of culture are carried out to the membrane with the most wrinkles and the yellowish fungus membranes are formed. The production process of high-titer PPD enriched with two antigens, namely ESAT-6 and CFP10, provided by the invention has high titer of PPD, and the contents of ESAT-6 and CFP10 are high. However, the product strain is a mycobacterium tuberculosis virulent strain CMCC93009(H37Rv), is a second type of pathogenic microorganism, has great threat to the health of production operators, has high safety protection level, has great potential threat to the environment, and is a local ecological disaster if a safety accident occurs.

With the development of science and technology and the improvement of safety requirements, the production conditions declared at the early stage of the strain can not meet the production requirements of GMP. The strain selection for manufacturing the tuberculin pure protein derivative is a development trend of the industry, and has low safety requirement, small threat to production operators and small influence on environment.

Disclosure of Invention

The invention aims to overcome the defects of a tuberculin pure protein derivative production process in the prior art (mainly defects of raw material strains), and applies a mycobacterium tuberculosis low-virulent strain CMCC93020(H37Ra) to a method for preparing the tuberculin pure protein derivative, and provides a matched preparation method according to the strain so as to overcome the defects of great harm to operators and environment, high requirements on production conditions and the like in the prior art (P3 production workshop), and finally obtains a protein which can be used for clinical diagnosis of tuberculosis, screening of a bacillus calmette-guerin inoculated object, monitoring of organism immune response after bacillus calmette-guerin inoculation and epidemiological monitoring.

In order to achieve the technical purpose, the following technical scheme is proposed:

a method for preparing tuberculin pure protein derivatives from a mycobacterium tuberculosis low virulent strain H37Ra comprises the following steps:

taking a mycobacterium tuberculosis low virulent strain CMCC93020(H37Ra) as a strain, and obtaining a bacterial liquid (containing bacterial metabolic protein) after amplification and inactivation;

salting out and precipitating the bacterial liquid, performing acid denaturation and precipitation, desalting and ultrafiltration, and performing sterilization and filtration to obtain a protein stock solution;

and (4) inspecting, diluting and packaging the protein stock solution to obtain a finished product of the tuberculin pure protein derivative.

Preferably, in the amplification, the amplification of the whole plant source solid culture medium is performed first, and then the amplification of the semi-solid culture medium is performed. Preparing a large batch of thalli in amplification of a full-plant-source solid culture medium, realizing large-scale production and preparing for subsequent batch finished products; in the semi-solid culture medium amplification, the metabolic protein of the thallus is convenient to collect, on one hand, the large-batch thallus formed in the full-plant-source solid culture medium amplification is connected, and on the other hand, the method is suitable for the preparation of subsequent protein stock solution and finished products;

in addition, the whole plant source solid culture medium is higher in safety than the animal source solid culture medium; compared with the traditional liquid culture medium, the semi-solid culture medium is adopted, the protein yield is high (30-40 mg/100mL of bacterial liquid for the semi-solid culture medium and less than 10mg/100mL of bacterial liquid for the traditional liquid culture medium), the culture time is short (about 4 weeks for the semi-solid culture medium and 8-10 weeks for the traditional liquid culture medium), the operation is simple (the bacterial liquid is directly inoculated on the potato blocks for the semi-solid culture medium, and the bacterial membrane passage is realized for the traditional liquid culture medium, the bacteria belong to young age, the passage time point is strict, the bacterial membrane is easy to sink and the like), the passage frequency is low, and the variation risk is low (the bacterial membrane passage in the traditional liquid culture medium is more than 2 generations).

Preferably, the whole plant source solid culture medium is a sutong potato soup solid culture medium, and the culture medium is 1000mL and comprises: 8.0g of sodium glutamate, 0.5g of dipotassium phosphate, 2.0g of citric acid, 0.5g of magnesium sulfate, 0.05g of ferric ammonium citrate, 60mL of glycerol, 940mL of potato soup and 20g of agar powder; wherein, the subculture proliferation inoculation amount of the Sutong potato soup solid culture medium is preferably not less than 2.5 mg/strain, and the culture time is preferably not less than 3 weeks;

the semi-solid culture medium is a potato Souton semi-solid culture medium, and comprises the following components in 1000 mL: 8.0g of sodium glutamate, 0.5g of dipotassium phosphate, 2.0g of citric acid, 0.5g of magnesium sulfate, 0.05g of ferric ammonium citrate, 60mL of glycerol and 940mL of water for injection. Subpackaging 500mL triangular bottles and 180mL bottles, and placing potato blocks in each bottle.

Preferably, in the salting-out precipitation, the inactivated bacteria liquid is firstly salted out and precipitated by ammonium sulfate, and then is subjected to trichloroacetic acid denaturation precipitation, so that the purity of the protein in the finished product can be ensured to be within 0.04mg (based on the total amount of polysaccharide and nucleic acid in each 1mg of tuberculin pure protein derivative); on the contrary, the purity of the protein in the finished product is only up to 0.09mg (the total amount of polysaccharide and nucleic acid contained in each 1mg of tuberculin pure protein derivative is up to 0.1mg in the current edition of Chinese pharmacopoeia);

wherein, ammonium sulfate is salted out and precipitated for 1-3 times, the final saturation of the ammonium sulfate is 40%, and the ammonium sulfate is salted out and precipitated for 2-4h, so that the efficiency and quality of protein salting out and precipitation are ensured;

the trichloroacetic acid denaturation precipitation is carried out for 3-5 times, the concentration of the trichloroacetic acid denaturation precipitation is 3-4%, and the trichloroacetic acid denaturation precipitation is carried out for 1-2h, so that the efficiency and the quality of the proteinic acid denaturation precipitation are ensured.

Preferably, during the acid-denatured precipitation, trichloroacetic acid is adopted to wash the polysaccharide impurities in the bacteria liquid by a wet method.

Preferably, in the desalting ultrafiltration, an ultrafiltration device with the molecular weight cutoff of 3KD is adopted for desalting, so that ammonium sulfate and trichloroacetic acid residues introduced in the precipitate are removed, and the protein purity in the finished product is improved.

Preferably, in the sterile filtration, a sterile filtration device with the pore diameter of 0.2 μm is adopted, and the precision of the sterile filtration device is further limited, so that the protein purity in a finished product is effectively improved.

Preferably, the finished tuberculin pure protein derivative is subpackaged by penicillin bottles and pre-filling and sealing modes.

Preferably, in the dilution, 0.01mol/L PBS solution with pH7.2-7.4 is used, and the PBS solution comprises 0.0005% polysorbate-80. In a conventional dilution process, a PBS solution containing 3.0g/L phenol, which is a carcinogen of three classes, and 0.0005% polysorbate-80 was used, which increases safety risks.

In the technical scheme, the prepared tuberculin pure protein derivative is multi-component composite protein, and the total amount of polysaccharide and nucleic acid contained in each 1mg of tuberculin pure protein derivative is less than 0.05 mg. The results of delayed hypersensitivity reactions of 24H and 48H caused by the mycobacterium tuberculosis attenuated strain CMCC93020(H37Ra) and the guinea pig sensitized by bacillus calmette-guerin are positive. Therefore, the tuberculin pure protein derivative can be used for clinical diagnosis of tuberculosis, screening of BCG vaccine inoculation objects, monitoring of organism immune reaction after BCG vaccine inoculation and epidemiological monitoring.

The first mention of PPD by seibert in 1928 was made and PPD prepared in this way in the us in 1944 was designated by the WHO as the mammalian international standard tuberculin. In 1955, the WHO and Union national Children's Foundation (UNICEF) mandate Danish serum institute to produce a large amount of PPD product, named PPD-RT23, which is the most widely used PPD product worldwide. The strain used in this product is a clinical isolate from tuberculosis patients, i.e. H37 Rv.

The Chinese tuberculin pure protein derivative (TB-PPD) was successfully developed by the Chinese medicine biological product assay in 1989, and the strain CMCC93009(H37Rv) is adopted, which is a traditional method of the world health organization. So far, no research has been found on the production of pure tuberculin protein derivatives by using a Mycobacterium tuberculosis low virulent strain H37Ra as a strain in a unit or an individual. However, with the improvement of GMP requirements, the production requirements of highly pathogenic and high-risk pathogenic microorganisms are higher and higher, the production cost is greatly improved, and even the production is forced to be stopped. Low pathogenic, low risk pathogenic microorganisms are an industry trend.

According to GMP requirements, BSL-3 protection grade is required for the second category pathogenic microorganisms, and the protection grade has high construction cost and expensive operation and maintenance cost; at present, only one unique new corona vaccine in China is the BSL-3 protection level. The three types of pathogenic microorganisms need BSL-2 protection level, belong to the conventional requirements of biological pharmacy, and have low construction, operation and maintenance cost.

In addition, the amplification of the strain full-plant-source solid culture medium and the amplification of the semi-solid culture medium are operated in a biological safety cabinet, but most of leaked thalli are taken out of a production place by a ventilation system, and cultures in the biological safety cabinet need to be moved to an incubator for culture and other bacteria-carrying wastes need to be moved to a sterilization cabinet for sterilization; these operations inevitably bring part of the bacteria to the production environment, posing a certain threat to the safety of the operators. The third category of pathogenic microorganisms has little threat to operators, while the second category of pathogenic microorganisms has great threat to operators; thalli in a production place is trapped by a ventilation system through a high-efficiency filter and then discharged out of the environment, but once an accident or poor management occurs in the high-efficiency filter, the thalli can be leaked to the natural environment, and a local ecological disaster is caused.

By adopting the technical scheme, the beneficial technical effects brought are as follows:

1) in the invention, the method for preparing the tuberculin pure protein derivative by using the mycobacterium tuberculosis low virulent strain CMCC93020(H37Ra) as a strain has low harm to operators and the environment, low requirements on the safety level of the most production workshop and low production cost;

2) in the invention, the whole production adopts a plant source culture medium, and is not influenced by animal infectious diseases (such as: the culture medium of the Roche eggs uses fresh eggs as a main nutrient source of bacteria, but when avian influenza or fowl plague occurs, the source of the eggs is uncontrollable, which greatly influences the amplification of strains, the subsequent product quality and the production efficiency), reduces the safety risk, and,

the production efficiency and the product quality are improved;

3) in the invention, the preparation time of the tuberculin pure protein derivative is shortened by controlling the conditions of salting-out precipitation of bacteria liquid, sterilization, filtration and the like, and the specific preparation time is controlled within 5d, so that the risk of bacterial contamination in the preparation process is indirectly reduced;

4) in the present invention, in the process of preparing the finished product from the protein stock solution, three carcinogens (such as: phenol preservatives), reducing safety risks;

5) in the invention, the tuberculin pure protein derivative finished product is packaged by penicillin bottles and pre-encapsulation, so that the safety risk in post-treatment is reduced.

Detailed Description

In the following, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples, the mycobacterium tuberculosis low virulent strain CMCC93020 is derived from China center for medical bacteria collection.

In the following examples, the culture medium involved comprises:

sutong medium, in 1000mL, comprising: 8.0g of sodium glutamate, 0.5g of dipotassium phosphate, 2.0g of citric acid, 0.5g of magnesium sulfate, 0.05g of ferric ammonium citrate, 60mL of glycerol and 940mL of water for injection;

suntong potato soup solid (slant) medium, 1000mL, comprising: 8.0g of sodium glutamate, 0.5g of dipotassium phosphate, 2.0g of citric acid, 0.5g of magnesium sulfate, 0.05g of ferric ammonium citrate, 60mL of glycerol, 940mL of potato soup and 20g of agar powder;

potato Souton semisolid culture medium, 1000mL, comprising: 8.0g of sodium glutamate, 0.5g of dipotassium phosphate, 2.0g of citric acid, 0.5g of magnesium sulfate, 0.05g of ferric ammonium citrate, 60mL of glycerol and 940mL of water for injection; subpackaging 500mL triangular bottles and 180mL bottles, and placing potato blocks (wherein, for the potato blocks, the potato blocks are cut into cubes with end surfaces of 25 × 25mm by using a special tool);

all media were autoclaved at 121 ℃ for 30min after preparation.

Example 1

A. seed batch resuscitation

Confirming that the appearance of a mycobacterium tuberculosis low-virulent strain CMCC93020 strain tube is not damaged, starting a seed batch, diluting the strain to the concentration of 10mg/mL by using a Souton culture medium, and uniformly mixing; adopting a Sutong potato soup solid slant culture medium, and paving the whole culture medium slant with the inoculation amount of 2.5 mg/piece; culturing at 36-38 deg.C for three weeks to obtain seed culture;

B. seed batch amplification

Taking a seed batch resuscitation culture, adding a threonine culture medium, eluting, grinding, centrifuging for 5min at the centrifugal force of 15000g, centrifuging, removing supernatant, weighing, and calculating the concentration of a bacterial liquid; adding a Sutong culture medium, and diluting the bacterial liquid to 100 mg/mL; adopting a Sutong potato soup solid slant culture medium, and paving the whole culture medium slant with the inoculation amount of 25 mg/piece; culturing at 36-38 deg.C for three weeks to obtain seed batch amplification culture;

C. bacterial liquid culture

Taking a seed batch amplification culture, adding a Sutong culture medium, eluting, grinding, centrifuging for 5min at the centrifugal force of 15000g, centrifuging, removing supernatant, weighing, and calculating the concentration of a bacterial liquid; adding a Sutong culture medium, and diluting the bacterial liquid to 150 mg/mL; adopting potato Sootong semi-solid culture medium, and spreading potato flour with inoculation amount of 150 mg/bottle; culturing at 36-38 deg.C for four weeks to obtain potato SUTONG semisolid culture;

D. autoclaving of bacteria liquid

Placing potato Soutong semisolid culture at 121 deg.C, and autoclaving for 20 min;

E. bacterial liquid collection

Taking the bacteria liquid after autoclaving, filtering and removing most of bacteria (such as double-layer silk fabric); centrifuging at a centrifugal force of 10000g for 10min, and collecting supernatant;

F. salting out and precipitating ammonium sulfate twice

Adding solid ammonium sulfate into the centrifuged bacterial liquid, completely dissolving until the final saturation degree is 40%, standing and precipitating for 2-4h, centrifuging for 30min at the centrifugal force of 10000g, and collecting the precipitate; dissolving the precipitate with PBS solution with pH7.3 and concentration of 10mmol/L, centrifuging at a centrifugal force of 10000g for 30min, and collecting the supernatant;

repeating the above operation for 1 time to obtain crude protein solution;

G. acid-denatured trichloroacetic acid precipitate for four times

Adding trichloroacetic acid solution with concentration of 40% into the crude protein solution after ammonium sulfate precipitation until the final concentration of trichloroacetic acid in the crude protein solution is 3-4%, standing for precipitation for 1-2 hr, centrifuging for 5min at centrifugal force of 5000g, and collecting precipitate; dissolving the precipitate with PBS (pH8.4) solution with concentration of 10mmol/L, centrifuging at a centrifugal force of 5000g for 5min, and collecting supernatant;

repeating the above operation for 2 times;

collecting the crude protein solution precipitated by trichloroacetic acid, adding trichloroacetic acid solution with concentration of 40% until the final concentration of trichloroacetic acid in the crude protein solution is 3-4%, standing for precipitation for 1-2 hr, centrifuging at centrifugal force of 5000g for 5min, and collecting precipitate; dissolving the precipitate in PBS (pH8.4) solution with concentration of 10mmol/L, and adding 1M sodium hydroxide solution to adjust pH to 8.0; centrifuging at a centrifugal force of 5000g for 5min, and collecting supernatant;

H. desalination by ultrafiltration

Taking the crude protein liquid precipitated by trichloroacetic acid, desalting by 4 times, ultrafiltering, concentrating, and concentrating repeatedly for 7 times;

I. sterilizing filtration

Taking the desalted crude protein liquid, sterilizing and filtering the crude protein liquid by using a filter element with the specification of 2.5 inches and 0.2 mu m, and collecting filtrate to obtain tuberculin pure protein derivative stock solution;

G. preparation of semi-finished product

According to the result of stock solution standardization (50000IU/mg protein), the stock solution is diluted to 50IU/mL activity by PBS with the pH value of 7.3 and the concentration of 10 mmol/L;

K. subpackaging finished products

And subpackaging the semi-finished product into 2mL penicillin bottles, wherein each penicillin bottle is 1.1mL (10 parts), each penicillin bottle is pre-filled and sealed with 0.5mL, and each penicillin bottle is 0.2mL (1 part).

Example 2

Based on example 1, this example discusses the control conditions in the production process to further illustrate the present technical solution.

(1) Salting-out and precipitation time of ammonium sulfate

The ammonium sulfate salting-out precipitation times were set to be 2 hours, 4 hours, 6 hours, 12 hours and 18 hours, respectively, and the results obtained are shown in table 1 below, and were found to be: under different precipitation time limits, the weight difference of the collected precipitates is not large, so the precipitation time is selected to be 2-4h according to the efficiency of the preparation method;

TABLE 1

Settling time	Weight of insoluble matter (mg)
		2h	28.1
4h	27.2
		6h	29.3
12h	31.0
		18h	28.9

(2) Final saturation degree of salting-out precipitation by ammonium sulfate

The final saturation degrees of salting-out precipitation with ammonium sulfate were set to 40%, 60% and 80%, respectively, and the results obtained are shown in Table 2 below and are known: when the final saturation of ammonium sulfate salting-out precipitation is 40%, the purity of the purified protein is higher;

TABLE 2

Saturation of ammonium sulfate	40％	60％	80％
				Protein Activity	Meets the requirements	Meets the requirements	Meets the requirements
Purity of protein	96.88％	95.78％	95.15％
				mg/100mL bacterial liquid	45.603	49.660	55.454

(3) Number of salting-out and precipitation of ammonium sulfate

The ammonium sulfate salting-out times were set to 1 and 2, respectively, and the results are shown in the following Table 3 and are known: when the salting-out precipitation times of the ammonium sulfate are 1 time, the purity of the purified protein is higher;

TABLE 3

Number of times of precipitation	1 time of	2 times (one time)
			Protein Activity	Meets the requirements	Meets the requirements
Purity of protein	90.70％	91.97％
			mg/100mL bacterial liquid	58.736	58.621

(4) Trichloroacetic acid denaturation and precipitation time

The trichloroacetic acid denaturation and precipitation time was set to 1h, 2h, 3h and 4h, respectively, and the results are shown in the following table 4 and known as follows: under different precipitation time limits, the weight difference of the collected precipitates is not large, so that the precipitation time is selected to be 1-2h according to the efficiency of the preparation method;

TABLE 4

(5) Acid-denatured precipitated concentration of trichloroacetic acid

The trichloroacetic acid denatured precipitate concentrations were set at 2%, 3% and 4%, respectively, and the results obtained are shown in table 5 below and are known: when the concentration of the trichloroacetic acid denaturation precipitate is 3-4%, the yield and purity of the purified protein are higher;

TABLE 5

Trichloroacetic acid concentration	2％	3％	4％
				Protein Activity	Meets the requirements	Meets the requirements	Meets the requirements
Purity of protein	91.24％	91.79％	92.91％
				mg/100mL bacterial liquid	51.109	53.810	56.928

(6) Acid denaturation and precipitation times of trichloroacetic acid

The results obtained by setting the numbers of acid-denatured precipitation of trichloroacetic acid to 4, 5 and 6 times, respectively, are shown in the following Table 6 and are known: when the times of trichloroacetic acid denaturation and precipitation are 4 times, the yield and the purity of the purified protein are higher;

TABLE 6

Number of times of washing polysaccharide	4 times (twice)	5 times (twice)	6 times of
				Protein Activity	Meets the requirements	Meets the requirements	Meets the requirements
Purity of protein	93.10％	90.07％	91.45％
				mg/100mL bacterial liquid	55.749	54.936	53.078

(7) Molecular weight cut-off by ultrafiltration

The ultrafiltration cut-offs were set at 3kD, 10kD, 30kD and 50kD, and the results are shown in Table 7 below and are known: with the increase of ultrafiltration cut-off molecular weight, the protein loss rate is increased, the protein purity is not greatly different, but the protein purity meets the requirements of Chinese pharmacopoeia, and the ultrafiltration cut-off molecular weight is finally selected to be 3KD under the comprehensive condition of protein activity, protein purity and protein loss rate.

TABLE 7

Molecular weight cut-off by ultrafiltration	3KD	10KD	30KD	50KD
					Protein Activity	Meets the requirements	Meets the requirements	Meets the requirements	Meets the requirements
Purity of protein	94％	93％	93％	94％
					Rate of protein loss	5％	11％	30％	51％

Example 3

Based on examples 1-2, this example further illustrates the technical scheme by examining the purified protein derivatives of tuberculin obtained.

Appearance of tuberculin pure protein derivative stock solution

Yellowish to brownish clear liquid, without insoluble matter or impurities.

Secondly, purity of stock solution

Protein detection: detecting the protein content according to the current edition (general rule 0731 second method) of Chinese pharmacopoeia;

polysaccharide detection: detecting the content of polysaccharide according to the current edition (tuberculin pure protein derivative) of Chinese pharmacopoeia;

and (3) nucleic acid detection: detecting the content of nucleic acid according to the current edition (general rule 0401) of Chinese pharmacopoeia;

the results obtained are shown in table 8 below, knowing: the total amount of polysaccharide and nucleic acid contained in each 1mg of tuberculin pure protein derivative is not higher than 0.1 mg.

TABLE 8 purity test results of tuberculin pure protein derivative stock solution

Third, the potency of the stock solution

The titer of the stock solution was measured according to the current edition of the Chinese pharmacopoeia (tuberculin protein derivative), and the results are shown in the following Table 9: 50000IU/1mg protein.

TABLE 9 results of potency assay of purified protein derivatives of tuberculin stock solutions

Fourth, sterility testing

According to the sterility test of the current edition (general rule 1101) of the Chinese pharmacopoeia, the following results are obtained: the stock solution is sterile.

Five, no mycobacterial test

Weighing 1.0mL of the stock solution, respectively inoculating to 10 Roche eggs, culturing at 37 deg.C for 4 weeks without growth of mycobacteria.

Sixthly, sensitization effect test

The test group and the control group respectively adopt 3 guinea pigs with the weight of 300-; and 15 days after the 3 rd injection, 0.1mL of the product containing 500IU is injected into each guinea pig of the test group and the control group in an intradermal way, and the continuous observation is carried out for 3 days, thus obtaining: there was no significant difference in the responses of the two groups of animals.

In summary, the results are shown in tables 10-11:

TABLE 10 quality test results of stock solutions of tuberculin purified protein derivatives

TABLE 11 detection results of purified protein derivatives of tuberculin

Example 4

Based on examples 1-3, this example discusses that the purified protein derivatives of tuberculin induce delayed hypersensitivity to guinea pigs sensitized with Mycobacterium tuberculosis attenuated strain CMCC93020, and further illustrates the present technical scheme.

S1: guinea pig

Weight 300-.

S2: skin test

Tuberculin pure protein derivative skin test negative guinea pigs.

S3: sensitization source

A mycobacterium tuberculosis low virulent strain CMCC93020 allergen (50mg/mL) is taken to be sterilized for 20min at 115 ℃, and is mixed with equivalent volume of Freund incomplete adjuvant and fully emulsified (25 mg/mL).

S4: sensitization

0.1mL of allergen bacteria solution (25mg/mL) was injected into each side of the groin, and skin test was performed five weeks after sensitization.

S5: delayed hypersensitivity reaction

Removing hair on the back of a guinea pig, injecting 0.2mL of national institute standard (50IU/mL) of tuberculin pure protein derivatives, 0.2mL of commercial products (Beijing auspicious sign, 50IU/mL) and 0.2mL of products (50IU/mL) into the skin, respectively recording the transverse diameter and the longitudinal diameter of the induration of each part for 24h and 48h, calculating the total average diameter of 24h and 48h, and detecting the effect of inducing delayed hypersensitivity by the mycobacterium tuberculosis attenuated strain CMCC93020 through sensitization, thus obtaining: the product is basically identical to national standard products and commercial products in inducing delayed hypersensitivity.

In summary, the results are shown in Table 12 below:

TABLE 12 tuberculin pure protein derivative Mycobacterium tuberculosis attenuated strain CMCC93020 sensitized animal experiment result

Example 5

Based on examples 1-4, this example discusses the purified protein derivative of tuberculin against Bacillus Calmette-Guerin D₂PB302 sensitized guinea pigs induced delayed hypersensitivity as further illustrated by the present protocol.

Y1: guinea pig

Weight 300-.

Y2: skin test

Tuberculin pure protein derivative skin test negative guinea pigs.

Y3: sensitization source

Bacillus Calmette-Guerin D₂PB302 allergen (10mg/mL), and Bacillus Calmette-Guerin solution was diluted to 5mg/mL with 0.9% NaCl solution.

Y4: sensitization

0.5mL of allergen bacteria solution (5mg/mL) was injected into each side of the groin, and skin test was performed five weeks after sensitization.

Y5: delayed hypersensitivity reaction

Removing hair on the back of a guinea pig, injecting 0.2mL of national institute standard (50IU/mL) of tuberculin pure protein derivatives, 0.2mL of commercial products (Beijing auspicious sign, 50IU/mL) and 0.2mL of products (50IU/mL) into the skin, respectively recording the transverse diameter and the longitudinal diameter of the induration of each part for 24h and 48h, calculating the total average diameter of 24h and 48h, and detecting the effect of inducing delayed hypersensitivity by the mycobacterium tuberculosis attenuated strain CMCC93020 through sensitization, thus obtaining: compared with the national standard products and the commercial products, the product has higher sensitivity in inducing delayed hypersensitivity.

In summary, the results are shown in Table 13 below:

TABLE 13 purified protein derivative of tuberculosis bacilli D₂Experimental results of PB302 sensitized animals

Claims

1. A method for preparing tuberculin pure protein derivatives from a mycobacterium tuberculosis low virulent strain H37Ra, which comprises the following steps: taking a mycobacterium tuberculosis low virulent strain CMCC93020(H37Ra) as a strain, and obtaining a bacterial liquid containing metabolic protein after amplification and inactivation; salting out and precipitating the bacterial liquid, performing acid denaturation and precipitation, desalting and ultrafiltration, and performing sterilization and filtration to obtain a protein stock solution; inspecting, diluting and packaging the protein stock solution to obtain a finished product of the tuberculin pure protein derivative;

in the amplification, amplification is carried out in a full plant source solid culture medium firstly, and then amplification is carried out in a semi-solid culture medium;

the whole plant source solid culture medium is a Sutong potato soup solid culture medium, and comprises the following components in 1000 mL: 8.0g of sodium glutamate, 0.5g of dipotassium phosphate, 2.0g of citric acid, 0.5g of magnesium sulfate, 0.05g of ferric ammonium citrate, 60mL of glycerol, 940mL of potato soup and 20g of agar powder;

the semi-solid culture medium is a potato Souton semi-solid culture medium, and comprises the following components in 1000 mL: 8.0g of sodium glutamate, 0.5g of dipotassium phosphate, 2.0g of citric acid, 0.5g of magnesium sulfate, 0.05g of ferric ammonium citrate, 60mL of glycerol and 940mL of water for injection; subpackaging 500mL triangular bottles and 180mL bottles, and placing potato blocks in each bottle;

in the precipitation, salting out and precipitating the inactivated bacteria liquid by adopting ammonium sulfate, and then performing trichloroacetic acid denaturation and precipitation; wherein, ammonium sulfate is salted out and precipitated for 2 times, the final saturation degree of the ammonium sulfate is 40 percent, and the ammonium sulfate is salted out and precipitated for 2 to 4 hours; the trichloroacetic acid denaturation precipitation is carried out for 4 times, the concentration of the trichloroacetic acid denaturation precipitation is 3-4%, and the trichloroacetic acid denaturation precipitation is carried out for 1-2 h.

2. The method for preparing purified protein derivatives of tuberculin with attenuated strain H37Ra of Mycobacterium tuberculosis as claimed in claim 1, wherein the acid-denatured precipitate is washed with trichloroacetic acid by wet method to remove the polysaccharide impurities.

3. The method for preparing tuberculin purified protein derivatives with Mycobacterium tuberculosis low virulent strain H37Ra as claimed in claim 1, wherein an ultrafiltration device with a molecular weight cut-off of 3KD is used in the desalting ultrafiltration.

4. The method for preparing tuberculin purified protein derivatives with the Mycobacterium tuberculosis low virulent strain H37Ra as claimed in claim 1 or 3, wherein a sterile filtration equipment with a pore size of 0.2 μm is used in the sterile filtration.

5. The method for preparing tuberculin purified protein derivatives with Mycobacterium tuberculosis low virulent strain H37Ra as claimed in claim 1, wherein in the dilution, PBS solution with pH of 7.2-7.4 and 0.01mol/L is used, and the PBS solution contains 0.0005% polysorbate-80.