CN101157949A - Fermentation amplification technique of engineering bacterium for producing anti-cancer drug TAT(m)-Survivin(T34A) - Google Patents

Abstract

The invention provides a fermentation and amplification technique of engineering bacteria for producing anticancer medicine of TAT (m)-Survivin (T34A). The technique comprises the following steps: A) the seed fluid is inoculated to the fermentation fluid for fermenting, with the fermenting temperature of 35-38 DEG C, pH6.5-7.5, ventilation quantity of 2-5ml/min, 5-80 percent of concentration of the growth period of dissolved oxygen; B) after 5 to 6 hours, the glucose solution is added in a permanent pH method and the thalli concentration is induced to 3-5g/L; C) the inorganic ammonia salt is added, and then IPTG is added and the glucose is added at the same time, and the concentration of carbon source in the fermentation tank is maintained at zero and the dissolving oxygen concentration at 40-60 percent, then, the thalli is incubated for 4 to 5 hours and then is collected. The fermentation and amplification technique of the invention has low producing cost and short producing period which is 10 to 12 hours. Meanwhile, the technique enhances the targeted protein expression level of unit bacteria, namely realizes the high expression of the targeted protein at the same time of enhancing the thalli density.

Description

Produce the fermentation amplification technique of the engineering bacteria of cancer therapy drug TAT (m)-Survivin (T34A)

Technical field

The invention belongs to bioengineering field, specifically, relate to the fermentation amplification technique of the engineering bacteria of a kind of production cancer therapy drug TAT (m)-Survivin (T34A).

Background technology

In application number is 200510026847.8 Chinese invention patent application, the contriver has made up fusion rotein TAT (m)-Survivin (T34A), and provide the shake flask fermentation method of this fusion rotein, this albumen is behind the escherichia coli expression purifying, can effectively import target cell, and then kill and wound target cell, the growth of B-CAP-37 and Hela cell is had remarkable inhibiting activity.Therefore, this recombinant protein medicine than gene therapy means safety, directly, fast, is easy to industrialization production, has the value and the good industrial prospect of further research and development.

High density fermentation is the important technology of modern genetic engineering product scale operation.Adopt the high density fermentation technology, improve the fermentation density of thalline, finally improve the specific production rate of product, not only can reduce volume of culture, optimize the downstream separation extraction, can also shorten the production cycle, reduce production costs, thereby greatly improve competitive power on market.The most frequently used and effective means is exactly the fed batch cultivation method, yet people tend to run into expression efficiency to be difficult to be improved to greatest extent and problems such as productive rate is lower, promptly along with the increase of cell density, the target protein expression amount obviously descends, and unit thalline expression amount significantly reduces.

Therefore, be necessary optimization for fermentation technology, under the situation of amplifying fermentation, can improve the target protein expression amount of unit thalline, when improving cell density, improve the Recombinant Protein Expression level, make it be easier to industrialization production.

Summary of the invention

The objective of the invention is to, the fermentation amplification technique of the engineering bacteria of a kind of production cancer therapy drug TAT (m)-Survivin (T34A) is provided, to adapt to the demand that fermentation is amplified.

The method that provides of the present invention may further comprise the steps:

A) seed liquor is inoculated in fermentation culture in the fermented liquid, culture temperature is 35-38 ℃, and pH is 6.5-7.5, and air flow is 2-5ml/min, and vegetative period, dissolved oxygen concentration was controlled at 5-80%;

B) after cultivating 5-6h, add glucose solution with permanent pH method and cultivate, treat to begin when cell concentration reaches 3-5g/L to induce;

When C) inducing, at first add inorganic ammonia salt, add IPTG then, mend glucose solution simultaneously, keep that carbon source concentration is 0 in the fermentor tank, dissolved oxygen concentration is controlled at 40-60%, cultivates 4-5h, collects thalline.

The fermentation amplification technique of the engineering bacteria of production cancer therapy drug TAT of the present invention (m)-Survivin (T34A), its low production cost, with short production cycle, only need 10-12h, simultaneously, also improve the target protein expression amount of unit thalline, promptly when cell density improves, realized the high expression level of target protein.

Description of drawings

Fig. 1 is the protein electrophoresis collection of illustrative plates of embodiment 4, and wherein, swimming lane M is marker, and swimming lane 1 is the total protein detected result when not inducing, and 2-5 is respectively and induces back 1,2,4, the total protein detected result of 5h.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples only are used to the present invention is described but not are used to limit scope of the present invention.

In following examples, employed fermentative medium formula is as follows:

Peptone 10g/L, yeast powder are 5g/L, and sodium-chlor is 5g/L, and glucose is 5g/L, penbritin 100mg/L.

In following examples, the detection method of cell concentration is as follows:

By measuring nutrient solution or its diluent absorbancy OD at wavelength 600nm place ₆₀₀Determine cell concentration, wherein, an OD ₆₀₀Unit is equivalent to 0.438g/L thalline (dry weight) concentration.

The detection method of target protein expression amount is as follows:

With the engineering bacteria induced product, add 2 sample damping fluids extraordinarily, through boil, centrifugal after, carry out the SDS-PAGE electrophoresis, concentrate glue 4%, separation gel 15%, coomassie brilliant blue R250 dyeing utilizes the expression level of Smartview (Bio-Rad) software analysis target protein.

In following examples, engineering bacteria E.coli BL21 (the DE3)/pRSET-B-tatm-survivin (T34A) that is used for fermentative production TAT (m)-Survivin (T34A) is 200510026847.8 the disclosed method structure of Chinese invention patent application acquisition according to application number.

In embodiment 3-5, described permanent pH method is meant glucose is added as sour incoming flow, promptly when pH is higher than set(ting)value, adds the method for glucose.

Embodiment 1, seed liquor preparation

Get engineering bacteria E.coli BL21 (DE3)/pRSET-B-tatm-survivin (T34A), insert in the 100mL LB liquid nutrient medium, in 250rpm, 37 ℃, cultivate 10h, make seed liquor.

Embodiment 2, TAT (m)-Survivin (T34A) engineering bacteria fermentation culture (comparative examples)

According to application number embodiment 4 disclosed methods in 200510026847.8 the Chinese patent application, fermentation E.coliBL21 (DE3)/pRSET-B-tatm-survivin (T34A) expresses TAT (m)-Survivin (T34A), and detecting thalline final concentration and target protein expression amount, the result is as shown in table 4.

Embodiment 3-5, TAT (m)-Survivin (T34A) engineering bacteria fermentation culture

Adopt fermentation amplification technique fermentation E.coli BL21 (DE3)/pRSET-B-tatm-survivin (T34A) of the present invention to express TAT (m)-Survivin (T34A), detailed process is as follows:

1) get the seed liquor that embodiment 1 obtains, the fermentation cylinder for fermentation that is inoculated in 3.7L, 5L and 30L by the inoculum size of 2-8% is respectively cultivated, and culture temperature is 35-38 ℃, pH is 6.5-7.5, air flow is 2-5ml/min, and vegetative period, dissolved oxygen concentration was controlled at 5-80%, and concrete parameter is as shown in table 1.

Table 1

Embodiment	Inoculum size (%)	Culture temperature (℃)	pH	Air flow (ml/min)	Dissolved oxygen concentration (%)
						3	5	37	7.0	4	80
4	2	35	7.5	2	30
						5	8	38	6.5	5	5

2) after cultivating 5-6h, add the 200-500g/L glucose solution with permanent pH method, every the 1h sampling, detect cell concentration, to treat to begin when cell concentration reaches 3-5g/L to induce, concrete parameter is as shown in table 2.

Table 2

Embodiment	Incubation time (h)	Constant pH	Glucose solution concentration (g/L)	Cell concentration (g/L)
					3	5.5	7.0	300	3.5
4	6	7.0	200	3
					5	5	7.0	500	5

When 3) inducing, at first add ammonium sulfate, add the IPTG of 0.25mM then every 1h, add altogether three times, each 1.5ml, mend into glucose with the constant flow velocity simultaneously, keep that carbon source concentration is 0 in the fermentor tank, dissolved oxygen concentration is controlled at 40-60%, concrete parameter sees Table 3, respectively at inducing preceding and inducing back 1,2,4,5h sampling, detect purity of protein, Fig. 1 is the detected result of embodiment 4.

Table 3

Embodiment	Inorganic ammonia salt (mM)	Flow velocity (g/h)	Dissolved oxygen concentration (%)
				3	380	3	50
4	750	5	40
				5	600	3.8	60

After inducing 4-5h, fermented liquid in the centrifugal 5-10min of 6000-10000rpm, is collected thalline, weighing thalline weight, and the proteic expression amount of testing goal, the result is as shown in table 4.

Table 4

Embodiment	Thalline final concentration g/L	Thalline wet weight (g/L)	Target protein expression amount (%)
				2	2.8	14	37
3	7	37	32
				4	5.8	35	30
5	9	41	35

According to The above results, compare with ordinary method, adopt TAT (m)-Survivin (T34A) the thalline final concentration of method fermentative production of the present invention to improve 2-3 doubly, though the target protein expression amount slightly reduces, the absolute yield of inclusion body protein improves greatly; And adopting present method fermentation period is 10-12h, and the ordinary method fermentation period also needs 9-10h, and both are very nearly the same, have therefore improved production efficiency greatly.

In sum, adopt preparation technology provided by the invention to produce TAT (m)-Survivin (T34A), simple to operate, fermentation costs is cheap, and good reproducibility when cell density improves, has also been realized the target protein high expression level.

Claims (6)

1. a fermentation amplification technique of producing the engineering bacteria of cancer therapy drug TAT (m)-Survivin (T34A) is characterized in that, may further comprise the steps:

A) seed liquor is inoculated in fermentation culture in the fermented liquid, culture temperature is 35-38 ℃, and pH is 6.5-7.5, and air flow is 2-5ml/min, and vegetative period, dissolved oxygen concentration was controlled at 5-80%;

B) after cultivating 5-6h, add glucose solution with permanent pH method and cultivate, treat to begin when cell concentration reaches 3-5g/L to induce;

When C) inducing, at first add inorganic ammonia salt, add IPTG then, mend glucose simultaneously, keep that carbon source concentration is 0 in the fermentor tank, dissolved oxygen concentration is controlled at 40-60%, cultivates 4-5h, collects thalline.

2. technology as claimed in claim 1 is characterized in that, described seed liquor is pressed the inoculum size inoculation of 2-8%.

3. technology as claimed in claim 1 is characterized in that, the concentration of glucose solution described in the step B is 200-500g/L.

4. technology as claimed in claim 1 is characterized in that, described inorganic ammonia salt is ammonium sulfate.

5. technology as claimed in claim 1 is characterized in that, described IPTG concentration is 0.75mM.

6. technology as claimed in claim 1 is characterized in that, described IPTG is that discontinuity adds.

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