CN112391431A - Fermentation medium and fermentation method of recombinant leukocyte inhibitory factor and hirulog peptide chimeric protein - Google Patents

Fermentation medium and fermentation method of recombinant leukocyte inhibitory factor and hirulog peptide chimeric protein Download PDF

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CN112391431A
CN112391431A CN201910763353.XA CN201910763353A CN112391431A CN 112391431 A CN112391431 A CN 112391431A CN 201910763353 A CN201910763353 A CN 201910763353A CN 112391431 A CN112391431 A CN 112391431A
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张贵民
冀成法
邵明学
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Lunan Pharmaceutical Group Corp
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Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation medium and a fermentation method for a recombinant leukocyte inhibitory factor and hirulog peptide chimeric protein. According to the invention, a basic culture medium and a supplementary culture medium are optimized, a fermentation culture method is changed, no carbon source is added in the initial fermentation stage for culture, and a carbon source is added after a certain period of culture, so that the growth of thalli is controlled. Stably realizes the high-density fermentation of the recombinant leukocyte inhibitory factor and the hirudin chimeric protein. Compared with low-density fermentation, the method has the advantages that the bacteria density is improved by 3-4 times, the yield of the fermented inclusion body is up to 23.8g/L and is improved by more than 5 times, the plasmid loss rate is low, the acetic acid production amount is low, the TNHH production efficiency is improved, and the method has good industrial application prospects.

Description

Fermentation medium and fermentation method of recombinant leukocyte inhibitory factor and hirulog peptide chimeric protein
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation medium and a fermentation method for a recombinant leukocyte inhibitory factor and hirulog peptide chimeric protein.
Background
Cerebrovascular diseases are one of three causes of death of human diseases at present, have the characteristics of high disability rate and high fatality rate, and seriously harm human health, wherein ischemic cerebrovascular diseases (ICVD) are more common clinically, account for about 60-70% of all cerebrovascular patients, can be divided into two categories of global cerebral ischemia and focal cerebral ischemia, and the pathogenesis of the cerebrovascular diseases is not completely clear. Studies have shown that cerebral edema and transient inflammatory responses are the main causes of cerebral ischemia and post-ischemic reperfusion injury. After cerebral ischemia, the combination of corresponding receptors on cell membranes after local massive thrombin activation is one of the main reasons for causing cerebral edema; a large number of neutrophils are infiltrated into the brain parenchyma after being gathered in local micro-blood vessels, and can also initiate inflammatory cascade reaction by generating active oxygen free radicals, cytokines, lipid metabolites, proteolytic enzymes and the like, wherein the inflammatory reaction plays a key role in secondary brain injury caused by acute cerebral ischemia, particularly reperfusion. Therefore, the active prevention and treatment of cerebral edema and excessive inflammatory reaction can effectively reduce the fatality rate of acute ischemic cerebrovascular disease and improve prognosis.
The recombinant leukocyte inhibitor and hirudin chimeric protein (TNHH) is prepared by recombining a neutrophil inhibitor (NIF, characterized by NIF-Gly (5-10)) and hirudin (hirulog) through genetic engineering, expressing the recombinant leukocyte inhibitor and the hirudin chimeric protein by escherichia coli, and then renaturing and purifying. TNHH consists of 283 amino acids, NIF 257 amino acids at the N-terminus, hirudin 20 peptide (Hirulog) at the C-terminus, and 5 glycine-linked regions in the middle, wherein the first amino acid at the N-terminus is methionine (Met). The NIF region in TNHH is completely inhibited except for increasing Met at position 1 compared with natural NIF, wherein Hirulog peptide (Hirul region) has two amino acid differences with Hirul reported in the literature on the market abroad, namely, the first D-proline is changed into L-type in the binding peptide FPRPGGGGGG, the 6 th glycine is changed into serine, and the specific structural formula of TNHH is shown as follows: Met-NIF (257) - (Gly) 5-FPRPGSGG-Hiruden (53-64). TNHH has a molecular weight of 31.5KD, pI4.5, contains 5 pairs of disulfide bonds, and is free of glycosylation.
TNHH is used as a novel protein medicine for treating acute cerebrovascular diseases, and is a novel anti-stroke bifunctional protein medicine for leukocyte activation and thrombin activity, which is designed mainly according to the pathological mechanism that local prothrombin is activated in the process of cerebral hemorrhage and cerebral thrombosis, and the microvascular obstruction and cerebral edema caused by platelets and brain tissue damage caused by affinity moistening and activating of leukocytes after thrombin formation. The medicine can be administered within 24 hours after acute onset for about 7 days, and can achieve multiple purposes of recovering damaged brain tissue, relieving cerebral edema and improving local microcirculation. Due to the good safety and obvious effectiveness of TNHH, the design of the double-target fusion protein realizes the purposes of reducing the risk of NIF and Hirul fragments and increasing the effectiveness. Therefore, TNHH is expected to be a brand new medicine which has obvious treatment and prevention effects (accelerating recovery and reducing sequelae) on acute stroke (particularly acute cerebral embolism) clinically. The method brings good news to the increasing number of the patients with the senile acute cerebrovascular accident, and simultaneously saves a great amount of medical and social costs.
In the prior art, the TNHH fermentation method is rarely reported, and the most important two problems in the fermentation problems of the genetic engineering bacteria are high-density fermentation of the bacteria and determination of induction conditions. The high-density fermentation technology is to improve the fermentation density of the cultured flora by changing the culture mode or culture conditions under a specific culture system, thereby improving the production of corresponding products. The culture method is based on the current advanced fermentation technology, utilizes various methods to improve the yield of the product, and can effectively improve the synthesis amount of related recombinant protein and reduce the culture volume when being applied to the fermentation culture process of the recombinant protein of the escherichia coli, and simultaneously can improve the purity of separation and extraction and greatly shorten the production period. Thus, the production cost is reduced from the side surface, and the production efficiency is improved.
The high-density culture technology greatly improves the yield and the productivity of the recombinant protein, reduces the fermentation cost and provides more space for the application of the recombinant escherichia coli. However, high level expression of foreign genes, not only related to the relationship among the host, vector and cloned gene, but also closely related to the environmental conditions, leads to insufficient oxygen supply and the production of large amounts of acetic acid in the culture medium, which greatly affects the growth of the bacterial cells. In addition, there is no correlation between the density of the cells and the expression level of the foreign protein, and the binding point between them is the determination of the induction conditions.
The recombinant plasmid of the genetic engineering bacteria has certain instability in the fermentation process, and the expected target gene product and yield cannot be obtained. The instability of plasmids is divided into structural instability of DNA fragments undergoing recombination, deletion or insertion and separation instability of plasmids not entering daughter cells during cell division. The stability of plasmids is influenced by a variety of genetic and environmental factors such as host and plasmid genotypes, host and plasmid interactions, the extent of gene expression, culture temperature, nutrient limitations, and the manner in which the reactor is operated. Genetic engineering fermentation usually requires high density culture to obtain more target products, however, too high cell density affects plasmid stability of the engineering bacteria.
Acetic acid is a main byproduct in the fermentation process of escherichia coli, and the accumulation of the acetic acid has obvious inhibition effect on the growth of thalli and the expression of products. Xuxinan et al, when culturing escherichia coli to express a native epidermal growth factor (hEGF), use glucose to replace acid liquor, and automatically add glucose solution to adjust without adding acid when the pH rises, but the adjustment has a certain lag, and the fluctuation of the sugar concentration and the accumulation of metabolic byproducts cannot be avoided.
The genetic engineering bacteria are fermented at high density, so that not only is higher thallus density required to be obtained, but also the accumulation of acetic acid, the stability of plasmids and high-efficiency expression are considered, and the four factors are mutually influenced and supplement each other.
The TNHH engineering bacteria also have the same problems in the fermentation process, such as low cell density, more acetic acid, high plasmid loss rate, low fermentation yield and the like. The defects are improved by adjusting a feeding method and controlling the feeding speed, and the effect is still poor, so a fermentation method of the recombinant leukocyte inhibitory factor and the leech peptide chimeric protein is needed to solve the technical problems.
Disclosure of Invention
In order to solve the problems of low TNHH fermentation density, high acetic acid production amount, high plasmid loss rate and low fermentation yield, the invention provides a fermentation culture medium and a fermentation method of a recombinant leukocyte inhibitory factor and hirulog chimeric protein.
The first purpose of the invention is to provide a basic culture medium for producing the chimeric protein of the recombinant leukocyte inhibitory factor and the hirudin, wherein the basic culture medium comprises glucose, yeast powder, tryptone, disodium hydrogen phosphate, potassium dihydrogen phosphate, ammonium chloride, sodium chloride, ferrous sulfate, magnesium sulfate and an antifoaming agent.
Preferably, the following components are included per liter of basal medium:
Figure BDA0002171081010000031
further, each liter of basal medium includes the following components:
Figure BDA0002171081010000032
the second purpose of the invention is to provide a supplemented medium for producing the recombinant chimeric protein of the leukocyte inhibitory factor and the hirudin, wherein the supplemented medium comprises yeast powder, tryptone and glucose.
Preferably, the supplementary culture medium is 30-50 g/L of yeast powder, 50-80 g/L of tryptone and 350-450 g/L of glucose.
The third purpose of the invention is to provide a fermentation method for producing the chimeric protein of the recombinant leukocyte inhibitory factor and the hirudin, which has the following specific technical scheme:
a fermentation method of a recombinant leukocyte inhibitory factor and hirulog peptide chimeric protein comprises the following steps:
(a) gradually amplifying the engineering bacteria in a seed culture medium to obtain a seed solution;
(b) inoculating the seed liquid into a basic culture medium of a fermentation tank for high-density culture, and feeding a supplemented culture medium in batches until the culture is finished.
Preferably, the engineering bacteria strain is pET-TNHH/BL21(DE3) PlysS, and the construction method thereof can refer to patent CN 03101155.1.
Preferably, the inoculation amount of the seed liquid inoculated into the basic culture medium of the fermentation tank is 5-10% of the volume of the basic culture medium.
Preferably, glucose in the fermentation basal medium needs to be sterilized separately and fed into the fermentation system after being fermented for 3-5 h.
Preferably, in the high-density culture process, the dissolved oxygen content of the fermentation liquor is controlled to be not less than 30%, the fermentation temperature is 37 +/-0.5 ℃, and the pH value is 6.5-7.0.
Preferably, the fed-batch medium is fed-batch medium fed-batch600Stopping feeding the culture medium for the first time when the temperature reaches 30-50 ℃.
Preferably, the second feeding of the feed medium refers to feeding of the rest of the feed medium after the addition of the inducer.
Preferably, during the feeding process, the feeding amount is 12-18% of the volume of the basal medium.
Preferably, the feeding time is 12-20 h in the feeding process.
Acetic acid is generated in the fermentation process, the generation of a large amount of acetic acid can reduce the pH value and inhibit the growth of thalli, the pH value of the fermentation system needs to be controlled in order to reduce the influence of the acetic acid on the fermentation, and preferably, the pH value of the fermentation system is controlled by ammonia water, and the amount of the ammonia water reflects the amount of the acetic acid generated in the fermentation process.
In a preferred embodiment, a fermentation method of the chimeric protein of the recombinant leukocyte inhibitory factor and the hirudin comprises the following steps:
(1) carrying out amplification culture on TNHH engineering bacteria in a seed culture medium step by step to obtain a seed solution;
(2) inoculating the seed liquid into an initial basal culture medium for fermentation culture;
(3) after culturing for 3-5 hours, slowly adding a glucose solution in a flowing manner, and continuously culturing;
(4) when the basic culture medium is exhausted, feeding a feed supplement culture medium to improve the thallus density;
(5) adding an inducer for induction culture;
(6) the feed medium was continuously fed until the end of the culture.
Preferably, the seed liquid culture step (1) is as follows:
inoculating engineering bacteria to the inclined plane of the solid seed culture medium, culturing at 37.0 ℃ for 13-18 h, taking out and storing at 2-8 ℃; selecting cultured slant colony of seed, inoculating in liquid seed culture medium, and culturing at 37.0 deg.C to OD in constant temperature oscillator6001.0-1.5 to obtain a first-stage shake flask seed solution; inoculating the cultured first-stage shake flask seed solution into liquid seed culture medium, and culturing at 37.0 deg.C to OD600And (5) 1.5-2.0 to obtain a second-stage shake flask seed solution.
The present invention is not limited to the seed liquid culture step, and any of the existing solid medium, liquid medium and culture method for seed liquid culture can be used in the present invention. In one embodiment, the solid seed medium consists of: 5g/L of yeast powder, 10g/L of tryptone, 10g/L of sodium chloride and 20g/L of agar powder; the liquid seed culture medium comprises the following components: 5g/L of yeast powder, 10g/L of tryptone and 10g/L of sodium chloride.
Preferably, the step (2) comprises the following specific steps: sterilizing the initial basal medium at 121 ℃ for 30 minutes; and (2) after sterilization, inoculating the seed solution obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH value to be 6.5-7.0, controlling the fermentation temperature to be 37 +/-0.5 ℃, and carrying out fermentation culture on dissolved oxygen of not less than 30%. The initial basal medium does not contain glucose components, and the glucose components are fed into a fermentation system after being sterilized separately.
Preferably, the step (3) comprises the following steps: dissolving glucose in appropriate amount of water, sterilizing at 115 deg.C for 30 min; and (3) after fermenting for 3-5 hours in the step (2), feeding glucose into the fermentation system, and controlling the feeding within 4-5 hours to be finished. Further preferably, the glucose feeding may be constant speed feeding or the feeding speed may be gradually increased.
Preferably, the step (4) comprises the following specific steps: when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise synchronously, the dissolved oxygen rises to more than 90 percent quickly, the supplemented culture medium begins to flow, the thallus density is sampled and detected at proper time, and when the thallus density OD is reached600Stopping feeding the supplemented medium when the temperature reaches 30-50 ℃.
Preferably, the step (5) comprises the following specific steps: and (4) after the feeding is suspended, synchronously increasing the dissolved oxygen and the pH of the fermentation system, adding an inducer when the dissolved oxygen rapidly rises to over 90 percent for induction culture, wherein the inducer is IPTG, and the final concentration of the inducer is 0.1-0.3 mM.
Preferably, the step (6) comprises the following specific steps: and (5) finishing the induction, and feeding the rest of the supplemented medium until the fermentation is finished.
Preferably, the total amount of the feed culture medium in the whole fermentation process is 12-18% of the volume of the fermentation basic culture medium;
preferably, the feeding time in the whole fermentation process is 12-20 h, and the feeding speed can be constant or gradually increased in the feeding process.
In a preferred embodiment, a fermentation method of the chimeric protein of the recombinant leukocyte inhibitory factor and the hirudin comprises the following steps:
(1) preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of tryptone, 10g/L of sodium chloride and 20g/L of agar powder;
liquid seed culture medium: 5g/L of yeast powder, 10g/L of tryptone and 10g/L of sodium chloride.
Inoculating engineering bacteria to the inclined plane of the solid seed culture medium, culturing for 13-18 h at 37.0 ℃, taking out and storing in a refrigerator at 2-8 ℃; selecting cultured slant colony of seed, inoculating in liquid seed culture medium, and culturing at 37.0 deg.C to OD in constant temperature oscillator6001.0-1.5 to obtain a first-stage shake flask seed solution; inoculating the cultured first-stage shake flask seed solution into liquid seed culture medium, and culturing at 37.0 deg.C to OD600And (5) 1.5-2.0 to obtain a second-stage shake flask seed solution.
(2) Sterilizing the initial basal medium without glucose at 121 ℃ for 30 minutes; and (2) after sterilization, inoculating the seed solution obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH value to be 6.5-7.0, controlling the fermentation temperature to be 37 +/-0.5 ℃, and carrying out fermentation culture on dissolved oxygen of not less than 30%.
The initial basal medium components were:
Figure BDA0002171081010000061
(3) dissolving glucose with the volume of 10-16 g/L of basic culture medium in a proper amount of water, and sterilizing for 30min at 115 ℃ for later use; after fermenting for 3-5 h in the step (2), feeding glucose, and controlling the feeding to be finished within 4-5 h; and (3) increasing the pH value in the flow addition process, and adjusting the pH value to 6.5-7.0 by using ammonia water.
(4) The supplementary culture medium comprises: 30-50 g/L yeast powder, 50-80 g/L tryptone and 350-450 g/L glucose.
Preparing a supplemented medium, and sterilizing at 121 ℃ for 30min for later use; when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, the supplemented medium begins to flow when the dissolved oxygen rises to more than 90 percent, the thallus density is sampled and detected timely, and when the thallus density OD is reached600Stopping feeding the supplemented medium when the temperature reaches 30-50 ℃.
(5) And (4) after the feeding is suspended, synchronously increasing the dissolved oxygen and the pH of the fermentation system, and adding an inducer IPTG with the final concentration of 0.1-0.3 mM for induction culture when the dissolved oxygen is increased to more than 90%.
(6) And (5) after the induction is finished, continuously feeding the residual supplementary culture medium, controlling the dissolved oxygen of the fermentation system to be 30-50% and the pH value to be 6.5-7.0, and continuously feeding the supplementary culture until the fermentation is finished.
The invention has the following beneficial technical effects:
the invention optimizes the basic culture medium and the supplementary culture medium, changes the fermentation culture method, does not add a carbon source for culture in the initial fermentation stage, and adds the carbon source after culturing for a certain time, so that the growth of the thalli is controlled. Stably realizes the high-density fermentation of the recombinant leukocyte inhibitory factor and the hirudin chimeric protein. By adopting the technical scheme of the invention, the fermentation density OD of TNHH600The method has the advantages that the strain density is improved by more than 75 times, compared with low-density fermentation, the strain density is improved by 3-4 times, the yield of the fermented inclusion body is up to 23.8g/L and is improved by more than 5 times, the plasmid loss rate is low, the acetic acid production amount is low, the production efficiency of TNHH is improved, and the method has good industrial application prospect.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be exemplary only, and are not intended to limit the scope of the invention, since numerous modifications will become apparent to those skilled in the art upon reading the present specification.
The species used in the following examples were constructed by themselves, with reference to patent CN 03101155.1. The reagents required in the fermentation process are commercially available without special mention.
Example 1
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, and cultured at 37 ℃ and 150r/min for 15h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the initial basal medium components according to the prescription amount, preparing 20L of the medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The initial basal medium components were:
Figure BDA0002171081010000071
Figure BDA0002171081010000081
(3) weighing 240g of glucose, adding the glucose into 400ml of water for dissolving, and sterilizing for 30min at 115 ℃ for later use; feeding glucose solution after culturing for 3.5 hours in the step (2), feeding at the flow rate of 2ml/min, and controlling the feeding for 4-5 hours; and (3) continuously increasing the pH value in the flow addition process, and adjusting the pH value to 6.5-7.0 by using ammonia water.
(4) Preparing 3L of supplemented medium: 40g/L of yeast powder, 65g/L of tryptone and 400g/L of glucose; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented culture medium begins to flow, the acceleration of the control flow is 2.5ml/min, the thallus density is sampled and detected in due time, and when the fed-batch is carried out for 4 hours, the thallus density OD is detected600Stopping feeding the feed medium when 34.86 is reached;
(5) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.2mM is added for induction culture;
(6) and (4) continuously feeding the rest of the fed-batch culture medium in the step (4) at a fed-batch speed of 2.5ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70% and controlling the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Example 2
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, cultured at 33 ℃ and 150 rpm to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 33 deg.C and 150r/min to OD600And culturing for 12h until the temperature reaches 1.5-2.0, and performing microscopic examination on the obtained product to obtain the TNHH fermentation seed liquid.
(2) Weighing the initial basal medium components according to the prescription amount, preparing 20L of the medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The initial basal medium components were:
Figure BDA0002171081010000091
(3) weighing 320g of glucose, adding 400ml of water for dissolving, and sterilizing for 30min at 115 ℃ for later use; and (3) feeding glucose solution after culturing for 3 hours in the step (2), feeding at a speed of 2ml/min, controlling the feeding for 4-5 hours, continuously increasing the pH value in the feeding process, and adjusting the pH value to 6.5-7.0 by using ammonia water.
(4) Preparing 3.6L of a supplemented medium: 30g/L of yeast powder, 80g/L of tryptone and 350g/L of glucose; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented culture medium begins to flow, the acceleration of the control flow is 2.0ml/min, the thallus density is sampled and detected in due time, and when the fed-batch is carried out for 4 hours, the thallus density OD is detected600When 35.25 is reached, stopping feeding the feed supplement culture medium;
(5) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.1mM is added for induction culture;
(6) and (4) continuously feeding the rest of the fed-batch culture medium in the step (4) at a fed-batch speed of 2.0ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70% and controlling the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Example 3
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, cultured at 35 ℃ at 150 rpm for 12h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 35 deg.C and 150r/min to OD600Culturing for 10h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the initial basal medium components according to the prescription amount, preparing 20L of the medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The initial basal medium components were:
Figure BDA0002171081010000101
(3) weighing 200g of glucose, adding 350ml of water for dissolving, and sterilizing for 30min at 115 ℃ for later use; feeding glucose solution after culturing for 5 hours, feeding at the flow rate of 1.8ml/min, and controlling the feeding for 4-5 hours; and (3) continuously increasing the pH value in the flow addition process, and adjusting the pH value to 6.5-7.0 by using ammonia water.
(4) Preparing 3L of supplemented medium: 50g/L of yeast powder, 50g/L of tryptone and 450g/L of glucose; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented culture medium begins to flow, the acceleration of the control flow is 2.0ml/min, the thallus density is sampled and detected in due time, and when the flow is added for 4.5 hours, the OD (OD) of the thallus density is detected600Stopping feeding the feed medium when 34.88 is reached;
(5) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.3mM is added for induction culture;
(6) and (4) continuously feeding the rest of the fed-batch culture medium in the step (4) at a fed-batch speed of 2.0ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70% and the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium for about 17 hours until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Example 4
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, cultured at 37 ℃ and 150 rpm to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the initial basal medium components according to the prescription amount, preparing 20L of the medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The initial basal medium components were:
Figure BDA0002171081010000111
(3) weighing 280g of glucose, adding 400ml of water for dissolving, and sterilizing for 30min at 115 ℃ for later use; feeding glucose solution after culturing for 4.5 hours, wherein the initial flow rate is 1ml/min, and finishing feeding at an increased speed for 4-5 hours; and (3) continuously increasing the pH value in the flow addition process, and adjusting the pH value to 6.5-7.0 by using ammonia water.
(4) Preparing 2.4L of a supplemented medium: 45g/L of yeast powder, 65g/L of tryptone and 450g/L of glucose; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented medium begins to flow, the acceleration of the control flow is 2.5ml/min, the thallus density is sampled and detected in due time, and when the fed-batch is carried out for 3 hours, the thallus density OD is detected600When 36.21 is reached, the feeding of the feed medium is stopped;
(5) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.2mM is added for induction culture;
(6) and (4) continuously feeding the rest of the fed-batch culture medium in the step (4) at a fed-batch speed of 2.5ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70% and controlling the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Example 5
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG to the concentration of 0.2mmol/L, inducing for 4h, centrifuging, collecting thallus, analyzing by SDS-PAGE electrophoresis, and screening to obtainSelecting an optimal colony streaking plate to culture at 37 ℃ for overnight when the TNHH single colony is obtained, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, and cultured at 37 ℃ and 150r/min for 15h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the initial basal medium components according to the prescription amount, preparing 20L of the medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The initial basal medium components were:
Figure BDA0002171081010000121
Figure BDA0002171081010000131
(3) weighing 240g of glucose, adding the glucose into 400ml of water for dissolving, and sterilizing for 30min at 115 ℃ for later use; and (3) feeding glucose solution after culturing for 2 hours in the step (2), controlling the feeding to be finished for 3 hours, feeding at the flow rate of 3ml/min, continuously increasing the pH value in the feeding process, and adjusting the pH value to be 6.5-7.0 by using ammonia water.
(4) Preparing 3L of supplemented medium: 40g/L of yeast powder, 65g/L of tryptone and 400g/L of glucose; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, and when the dissolved oxygen rises to more than 90 percent rapidlyFeeding the supplement culture medium at the beginning, controlling the flow acceleration at 2.0ml/min, sampling at proper time to detect the thallus density, and detecting the thallus density OD after feeding for 4h600When 34.57 is reached, the feeding of the feed medium is stopped;
(5) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.2mM is added for induction culture;
(6) and (4) continuously feeding the rest of the fed-batch culture medium in the step (4) at a fed-batch speed of 2.0ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70% and controlling the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Example 6
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, and cultured at 37 ℃ and 150r/min for 15h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the initial basal medium components according to the prescription amount, preparing 20L of the medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The initial basal medium components were:
Figure BDA0002171081010000141
(3) weighing 280g of glucose, adding 400ml of water for dissolving, and sterilizing for 30min at 115 ℃ for later use; feeding glucose solution after culturing for 6 hours, feeding at the flow rate of 2.0ml/min, and controlling the feeding for 4-5 hours; and (3) continuously increasing the pH value in the flow addition process, and adjusting the pH value to 6.5-7.0 by using ammonia water.
(4) Preparing 3.2L of a supplemented medium: 40g/L of yeast powder, 65g/L of tryptone and 400g/L of glucose; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented culture medium begins to flow, the acceleration of the control flow is 2.5ml/min, the thallus density is sampled and detected in due time, and when the fed-batch is carried out for 4 hours, the thallus density OD is detected600When 35.19 is reached, the feeding of the feed medium is stopped;
(5) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.2mM is added for induction culture;
(6) and (4) continuously feeding the rest of the fed-batch culture medium in the step (4) at a fed-batch speed of 2.5ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70% and controlling the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Example 7
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, and cultured at 37 ℃ and 150r/min for 15h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the initial basal medium components according to the prescription amount, preparing 20L of the medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The initial basal medium components were:
Figure BDA0002171081010000151
(3) weighing 240g of glucose, adding the glucose into 400ml of water for dissolving, and sterilizing for 30min at 115 ℃ for later use; feeding glucose solution after culturing for 5 hours, and controlling the feeding for 4-5 hours; and (3) adding at the flow rate of 2ml/min, continuously increasing the pH value in the adding process, and adjusting the pH value to 6.5-7.0 by using ammonia water.
(4) Preparing 4L of supplemented medium: 60g/L of yeast powder, 40g/L of tryptone and 300g/L of glucose; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented culture medium begins to flow, the acceleration of the control flow is 3.0ml/min, the thallus density is sampled and detected in due time, and when the fed-batch is carried out for 4 hours, the thallus density OD is detected600Stopping feeding the feed medium when the temperature reaches 32.56 ℃;
(5) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.2mM is added for induction culture;
(6) and (4) continuously feeding the rest of the fed-batch culture medium in the step (4) at a fed-batch speed of 3.0ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70% and controlling the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Example 8
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, and cultured at 37 ℃ and 150r/min for 15h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the basic culture medium components according to the prescription amount, preparing 20L of culture medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The basic culture medium comprises the following components:
Figure BDA0002171081010000161
(3) preparing 3L of supplemented medium: 40g/L of yeast powder, 65g/L of tryptone and 400g/L of glucose; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented culture medium begins to flow, the acceleration of the control flow is 2.5ml/min, the thallus density is sampled and detected in due time, and when the fed-batch is carried out for 4 hours, the thallus density OD is detected600Stopping feeding the culture medium when 33.83 is reached;
(4) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.2mM is added for induction culture;
(5) continuously feeding the rest of the fed-batch culture medium in the step (3) at a fed-batch speed of 2.5ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70 percent and keeping the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium for 18 hours until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Comparative example 1
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, and cultured at 37 ℃ and 150r/min for 15h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the initial basal medium components according to the prescription amount, preparing 20L of the medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The initial basal medium components were:
Figure BDA0002171081010000171
Figure BDA0002171081010000181
(3) weighing 320g of glycerol, and sterilizing at 121 ℃ for 30min for later use; feeding glycerol after culturing for 3.5 hours in the step (2), feeding at the flow rate of 1.2ml/min, and controlling the feeding for 4-5 hours; and (3) continuously increasing the pH value in the flow addition process, and adjusting the pH value to 6.5-7.0 by using ammonia water.
(4) Preparing 3L of supplemented medium: 40g/L of yeast powder, 65g/L of tryptone and 400g/L of glucose; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented culture medium begins to flow, the acceleration of the control flow is 2.5ml/min, the thallus density is sampled and detected in due time, and when the fed-batch is carried out for 4 hours, the thallus density OD is detected600When 35.84 is reached, stopping feeding the feed culture medium;
(5) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.2mM is added for induction culture;
(6) and (4) continuously feeding the rest of the fed-batch culture medium in the step (4) at a fed-batch speed of 2.5ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70% and the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium for 18 hours until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Comparative example 2
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. aAnd (3) level seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, and cultured at 37 ℃ and 150r/min for 15h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the initial basal medium components according to the prescription amount, preparing 20L of the medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The initial basal medium components were:
Figure BDA0002171081010000191
(3) sterilizing 320g of glycerol at 121 ℃ for 30min for later use; feeding glycerol after culturing for 3.5 hours in the step (2), feeding at the flow rate of 1.2ml/min, and controlling the feeding for 4-5 hours; and (3) continuously increasing the pH value in the flow addition process, and adjusting the pH value to 6.5-7.0 by using ammonia water.
(4) Preparing 3L of supplemented medium: 40g/L of yeast powder, 65g/L of tryptone and 400g/L of glycerol; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented culture medium begins to flow, the acceleration of the control flow is 2.5ml/min, the thallus density is sampled and detected in due time, and when the fed-batch is carried out for 4 hours, the thallus density OD is detected600When 36.82 is reached, the feeding of the feed medium is stopped;
(5) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.2mM is added for induction culture;
(6) and (4) continuously feeding the rest of the fed-batch culture medium in the step (4) at a fed-batch speed of 2.5ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70% and the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium for 18 hours until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Comparative example 3
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, and cultured at 37 ℃ and 150r/min for 15h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the basic culture medium components according to the prescription amount, preparing 20L of culture medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The basic culture medium comprises the following components:
Figure BDA0002171081010000201
(3) preparing 3L of supplemented medium: 40g/L of yeast powder, 65g/L of tryptone and 400g/L of glycerol; sterilizing at 121 deg.C for 30min, and cooling;
when the nutrients of the basic culture medium are exhausted in the fermentation process, the dissolved oxygen and the pH of the fermentation system rise simultaneously, when the dissolved oxygen rises to more than 90 percent rapidly, the supplemented culture medium begins to flow, the acceleration of the control flow is 2.5ml/min, the thallus density is sampled and detected in due time, and when the fed-batch is carried out for 4 hours, the thallus density OD is detected600When 36.51 is reached, the feeding of the feed medium is stopped;
(4) the dissolved oxygen and the pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, IPTG with the final concentration of 0.2mM is added for induction culture;
(5) continuously feeding the rest of the fed-batch culture medium in the step (3) at a fed-batch speed of 2.5ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70 percent and keeping the pH value to be 6.5-7.0, continuously feeding the fed-batch culture medium for 18 hours until the fed-batch culture is finished, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
Comparative example 4
(1) Preparing a seed solution:
solid seed culture medium: 5g/L of yeast powder, 10g/L of peptone, 10g/L of sodium chloride and 20g/L of agarose.
Liquid seed culture medium: 5g/L of yeast powder, 10g/L of peptone and 10g/L of sodium chloride.
a. And (3) screening strains: taking a strain pET-TNHH/BL21(DE3) PlysS, streaking and activating an LB plate, culturing at 37 ℃ overnight, selecting a single colony from the plate, inoculating the single colony in an 8ml LB liquid test tube, and culturing at 37 ℃ and 180r/min until OD is reached6001.0-1.5, adding IPTG (isopropyl thiogalactoside) to the concentration of 0.2mmol/L, inducing for 4 hours, centrifugally collecting thalli, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis, screening to obtain TNHH single colonies, selecting an optimal colony, streaking a flat plate, culturing at 37 ℃ for overnight, and storing at 4 ℃ after the culture is finished;
b. primary seed liquid culture: from the re-streaked plate, a single colony was picked and inoculated into a liquid test tube containing 8mL of seed medium, and cultured at 37 ℃ and 150r/min for 15h to OD6000.5-1.0, as first-class seed liquid;
c. secondary seed liquid culture: inoculating the primary seed solution at 1% inoculation ratio into 500mL triangular flask containing 250mL seed culture medium, culturing at 37 deg.C and 150r/min to OD600And culturing for 6h until the temperature reaches 1.5-2.0, and performing microscopic examination to obtain the seed liquid for TNHH fermentation.
(2) Weighing the basic culture medium components according to the prescription amount, preparing 20L of culture medium, and sterilizing for 30 minutes at 121 ℃; and (2) cooling the fermentation culture medium to 37 ℃ after sterilization, inoculating the seed liquid obtained in the step (1) into a fermentation tank according to the volume ratio of 5-10% of the inoculation amount, adjusting the pH to 6.5-7.0 by ammonia water or phosphoric acid, controlling the fermentation temperature to be 37 +/-0.5 ℃, introducing air at the ventilation ratio of 1:2, and adjusting the rotation speed to ensure that the dissolved oxygen is higher than 35% to perform fermentation culture.
The basic culture medium comprises the following components:
Figure BDA0002171081010000211
(3) preparing 2L of supplemented medium: 40g/L of yeast powder, 65g/L of tryptone and 400g/L of glycerol; sterilizing at 121 deg.C for 30min, and cooling;
(4) the dissolved oxygen and pH of the fermentation system rise synchronously, and when the dissolved oxygen rises to more than 90 percent, the density OD of the thallus is detected600At 11.37, IPTG was added to the final concentration of 0.2mM for induction culture;
(5) feeding the culture medium at a feeding speed of 2.5ml/min, controlling the temperature to be 37 +/-0.5 ℃, maintaining the dissolved oxygen at 30-70 percent and the pH value to be 6.5-7.0, continuing feeding the culture medium for 4 hours, and finishing the fermentation culture. And (3) carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the fermentation liquor to determine the TNHH protein expression quantity and the plasmid loss rate.
The results of fermentation broth assays of examples 1-8 and comparative examples 1-4 are shown in Table 1.
Table 1: EXAMPLES 1-8 AND COMPARATIVE EXAMPLES 1-4 fermentation broth test results
Figure BDA0002171081010000221

Claims (10)

1. A basic culture medium for producing the chimeric protein of the recombinant leukocyte inhibitory factor and the hirudin peptide is characterized by comprising the following components in every liter of the basic culture medium:
Figure FDA0002171080000000011
2. a feed medium for producing a chimeric protein of a recombinant leukocyte inhibitory factor and a hirudin peptide, which comprises the following components in percentage by weight: 30-50 g/L yeast powder, 50-80 g/L tryptone and 350-450 g/L glucose.
3. A fermentation method of a recombinant leukocyte inhibitory factor and hirulog peptide chimeric protein is characterized by comprising the following steps:
(a) gradually amplifying the engineering bacteria in a seed culture medium to obtain a seed solution;
(b) inoculating the seed liquid into a basic culture medium of a fermentation tank for high-density culture, and feeding a supplemented culture medium in batches until the culture is finished.
4. The fermentation process of claim 3, wherein the engineered bacterial species is pET-TNHH/BL21(DE3) PlysS.
5. The fermentation process of claim 3, wherein the seed solution is inoculated into the fermenter base medium in an amount of 5% to 10% by volume of the base medium.
6. The fermentation method according to claim 3, wherein the glucose in the basic culture medium is fed into the fermentation system after 3-5 hours of fermentation, and the feeding is controlled to be completed within 4-5 hours.
7. The fermentation method according to claim 3, wherein the dissolved oxygen in the fermentation broth is controlled to be not less than 30% during the high-density culture, the fermentation temperature is 37 ± 0.5 ℃, and the pH is 6.5-7.0.
8. The fermentation process of claim 3, wherein the total feed in the feed process is 12-18% of the basal medium volume.
9. The fermentation method according to claim 3, wherein the fed-batch culture medium is fed-batch culture medium fed-batch.
10. The fermentation method of claim 9, wherein the cell density is measured at the time when OD is reached during the first feeding of the feed medium600Stopping feeding the culture medium for the first time when the temperature reaches 30-50 ℃.
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