CN1360022A - Recombinant human epidermal growth factor and its preparing process and medicinal composition - Google Patents

Abstract

A recombinant human epidermal growth factor is prepared through configuring and cloning hEGF gene with the codon preferable to colibacillus, configuring and cloning alkaline phosphoresterase promotion gene and signal peptide sequence gene fragment phoA-P+sig, configuring expression plasmid pAE-8, converting host bacterium with pAE-8, screening to obtain engineering strain EE-8, culturing it in Lpi culture medium, and induced expression of rhEGF. Its advantages are high efficiency and stability in secretion and expression of rhEGF. The rhEGF medicine composition for treating digestive ulcer is also disclosed.

Description

Recombinant human epidermal growth factor, preparation method and pharmaceutical composition thereof

The invention belongs to the field of biomedicine, and particularly relates to a recombinant human epidermal growth factor (rhEGF) prepared by a recombinant DNA technology and a pharmaceutical composition for treating peptic ulcer by using the recombinant human epidermal growth factor.

Human Epidermal Growth Factor (Human Epidermal Growth Factor), abbreviated as hEGF, also known as β -urogastrin (B-urogastron), is a small protein without glycosyl and containing 53 amino acids, the molecular weight of which is about 6216 dalton, the isoelectric point is 4.7, the N-terminal of hEGF is asparagine (Asn), the C-terminal is arginine (Arg), and hEGF contains threepairs of disulfide bonds to maintain a stable protein configuration required for its biological activity.

Cohen and h.gregorg isolated human epidermal growth factor simultaneously from human urine in 1975. It is also called as gastric inhibitory hormone because of its gastric acid secretion resistance. hEGF is contained in body fluids such as urine, blood, saliva, tears, semen, milk, gastric juice and medullary fluid, wherein the contents of urine, milk and semen are high. The human body contains hEGF in many tissues and glands, such as thyroid gland, pancreas, salivary gland, jejunum, duodenum, and kidney, wherein the content of hEGF in salivary gland, pancreas, kidney, and thyroid is high.

Research shows that hEGF is a multifunctional important cytokine which can stimulate the growth of epidermal cells, endothelial cells and fibroblasts, thereby promoting the healing of burns, wounds and surgical wounds and accelerating the growth of transplanted epidermis; hEGF also promotes the growth of corneal epithelial cells, parenchymal cells, endothelial cells, and thus can be used to treat corneal trauma, corneal ulceration, chemical burns, and the like, and to accelerate the growth of transplanted corneas.

Although the primary structure of hEGF protein was elucidated as early as 1975 in Cohen, S., Carpentre, G. (Proc. Natl. Acad. Sci. USA, 1975, 72: 1317) and Gregory, H. (Nature, 1975, 257: 31), the nucleotide sequence of hEGF cDNA was not elucidated by researchers of Chiron corporation until 1986 (Graeme, I.B.et. et. Nucleic Acids Res., 1986, 14 (21): 8427).

Since hEGF has a wide range of biological functions and therapeutic effects, it is necessary to develop engineering bacteria capable of efficiently expressing rhEGF and a method for mass-producing rhEGF and a drug prepared using rhEGF in order to meet the needs of clinical treatment.

The investigators used their own synthetic alkaline phosphatase promoter (phoA), signal peptide sequence; the hEGF gene is synthesized by the self-designed codon, and the high-efficiency secretory expression plasmid is constructed in the multi-copy plasmid and is highly expressed in escherichia coli. In laboratory research, the hEGF secreted and expressed by engineering bacteria EE-8 or EET-8 in a shake flask can reach 30mg/L, and the hEGF expressed in a 15-liter fermentation tank can reach 60 mg/L. In a 150L fermentation tank, the secretion expression rhEGF reaches more than 100mg.

In view of the fact that the currently clinically commonly used drugs for treating peptic ulcer, namely Loxec (Omerazol) and Bayer gastric Daxi, have the function of stimulating the gastric mucosa to secrete EGF, hEGF can inhibit gastric acid, increase the blood flow of gastric mucosa, increase the synthesis of RNA, DNA and protein in gastric mucosal cells and promote the growth of mucosal cells, thereby promoting the healing of ulcer, improving the quality of ulcer healing and ensuring that the healed ulcer is not easy to relapse, the invention further develops the rhEGF preparation for treating the peptic ulcer by oral administration on the basis of efficiently expressing rhEGF.

Therefore, an object of the present invention is to provide an engineering bacterium capable of efficiently and stably expressing rhEGF.

It is another object of the present invention to provide a method for efficiently and stably preparing rhEGF.

It is another object of the present invention to provide a pharmaceutical composition for treating peptic ulcer.

The engineering strain EE-8 provided by the invention is an rhEGF expression strain which is preserved in China general microbiological culture Collection center and has thepreservation number of 02787-1.

The engineering strain EE-8 is obtained by transforming alkaline phosphatase-deleted Escherichia coli YK537 with an expression plasmid pAE-8.

The method for preparing the recombinant human epidermal growth factor comprises the following steps:

constructing and cloning the synthesized hEGF gene by using codon preferred by escherichia coli;

constructing and cloning an alkaline phosphatase promoter phoA-P and a signal peptide sequence sig;

cloning phoA-P and sig into a plasmid pTZ18R to construct an expression plasmid pAE-8;

transforming Escherichia coli YK537 by using expression plasmid pAE-8 and screening to obtain engineering strain EE-8;

culturing engineering bacteria EE-8 in an Lpi culture medium with controlled inorganic phosphorus content, and inducing and expressing rhEGF.

The pharmaceutical composition comprises a therapeutically effective amount of recombinant human epidermal growth factor and pharmaceutically acceptable excipients.

The present invention is described in detail below.

The present invention relates to recombinant human epidermal growth factor prepared by recombinant DNA technology. In particular to construction of an engineering strain EE-8 and a method for preparing a recombinant human epidermal growth factor by using the engineering strain EE-8. The invention also relates to a pharmaceutical composition for treating peptic ulcer prepared by the recombinant human epidermal growth factor and pharmacodynamic, toxicological and pharmacokinetic studies thereof.

Firstly, construction of engineering strains:

1. construction of expression plasmid:

1.1. construction and cloning of synthetic hEGF Gene

Native hEGF consists of 53 amino acids, the entire gene is 159 base pairs (fig. 1), and the design consists of 6 synthetic fragments. An EcoRI cut was added to the 5 'end of the gene, and a HindIII cut and two TAA terminators were added to the 3' end. The present inventors considered the following in synthesizing a gene: a, adopting codons preferred by escherichia coli; b, secondary structure formation is avoided in the fragment; c, avoiding repeated sequences among fragments as much as possible; d, using secretory expression, the hEGF gene has no ATG, the first code is AAT (Asn), which is identical to native hEGF (see FIG. 2A).

After the 1 st and 6 th fragments are phosphorylated, the 6 fragments are mixed by equal molecular weight, and are annealed together to be connected into an hEGF whole gene fragment, the multicopy pTZ18R plasmid is cut by EcoRI and HindIII, and then the recombinant ligation (shown in figure 3A) is carried out and JM103 receptor bacteria are transformed. The pEG-14 recombinant plasmid was selected by EcoRI, HindIII digestion and sequence analysis (see FIG. 4), and contained the synthetic hEGF gene as designed.

1.2. Construction and cloning of alkaline phosphatase promoter and signal peptide sequence

When designing the synthesis, we fully followed the natural sequence phoA-P + sig sequence of the E.coli alkaline phosphatase promoter gene phoA-P and the signal peptide sequence (sig). Consists of 8 synthetic fragments. PstI, BglII, XbaII, etc. cleavage sites were added to the 5' end of phoA-P to facilitate construction. An EcoRI cut was added to the 3' end of the sig sequence (see FIG. 2B).

After the 1 st and 8 th fragments are phosphorylated, 8 fragments are mixed and annealed by equimolar molecules and then are connected to obtain the phoA-P + sig gene fragment. The pTZ18R plasmid was digested with PstI and EcoRI, and then ligated with the ligated synthetic fragment by recombinant ligation, to transform JM 103. The pAP-2 recombinant plasmid was selected by digestion with PstI, EcoRI and sequence analysis, and had the same sequence as the native gene (see FIG. 3B).

1.3. Construction of expression plasmid pAE-8

The plasmid pAP-2 is cut by PstI and EcoRI to obtain phoA-P + sig fragment, the hEGF gene fragment obtained by cutting pEG-14 by EcoRI and HindIII is firstly connected with the plasmid pTZ18R cut by PstI and HindIII, and then JM103 is transformed. Expression plasmid pAE-8 (see FIG. 5) was selected by digestion with PstI, HindIII and sequence analysis.

1.3.1. Nucleotide sequence of target gene

The synthesized hEGF target gene on pAE-8 plasmid was subjected to nucleotide sequence analysis, and the determined nucleotide sequence is shown in FIG. 6 and is consistent with the hEGF sequence designed during synthesis. The hEGF gene has 159 nucleotides and codes 53 amino acids.

1.3.2. Expression plasmid restriction map

The constructed pAE-8 (see FIG. 5) secreting expression plasmids were characterized as follows:

(1) the promoters used were the alkaline phosphatase promoter (phoA) and the signal peptide sequence (sig) of E.coli synthesized by the inventors.

(2) For cloning convenience, a PstI isocleavage site was added to the 5 'end of the phoA promoter, and an EcoRI cleavage site was added to the 3' end after the signal peptide, which was about 190bp in length.

(3) The synthetic human epidermal growth factor (hEGF) gene is 159 nucleotides, and has EcoRI cut point added to the 5 'end and HindIII cut point added to the 3' end of hEGF gene for about 165 nucleotides.

FIG. 7 shows the restriction map of the constructed secretory expression plasmid pAE-8.

Wherein, the first column shows that pAE-8 is digested by PstI and EcoRI to obtain an about 190bp digested fragment containing a phoA promoter and a signal peptide sequence.

The second column indicates the molecular weight marker for pAE-8.

The third column shows that pAE-8 is digested by PstI and HindIII to obtain an about 355bp fragment containing a phoA promoter, a signal peptide sequence and an hEGF gene.

The fourth column shows that pAE-8 was digested with EcoRI and HindIII to give an about 165bp fragment containing the hEGF gene.

2. Construction and screening of genetically engineered bacteria

2.1. Construction of genetically engineered bacteria

In the selection of host bacteria, Escherichia coli YK537 deficient in alkaline phosphatase was used. YK537 belongs to K12 strain, and is a gram-negative bacterium.

The gene characteristics of the host bacterium YK 537:

F^-leuB6 thi hsdR hsdM lacy rpsL20 galk2 ara-14 xyl-5 mtl-1 supE44endl^-phoA8 recA1

phenotypic characteristics of host bacterium YK 537: the alkaline phosphatase gene is deleted, and the host bacterium can not express the alkaline phosphatase.

YK537 was transformed with expression plasmid pAE-8 to give expression strain EE-8.

2.2 screening of engineered strains

Selecting a plurality of transformed colonies, inoculating the transformed colonies into a high-phosphorus culture medium for overnight growth, centrifuging the next day, and then changing the low-phosphorus culture medium for induction. After induction, 1ml of bacterial liquid is taken, the bacterial body obtained after centrifugation is suspended in a solution containing lysozyme, the bacterial body is placed in an ice bath and then centrifuged, and the supernatant is taken for electrophoretic analysis. The EE-86 strain with the highest hEGF secretion expression amount is selected as a genetic engineering strain (abbreviated as EE-8).

Culturing and inducing expression of engineering bacteria:

the foreign scholars such as Oka et al (Wakunaga) generally put the engineering bacteria in a high phosphorus (640. mu.M KH)₂PO₄) Similar to M9 medium (TG-20 for short) overnight, in low phosphorus (32. mu.M KH)₂PO₄) M9-like medium (TG-1 for short) is used for inducing expression. Under the conditions of culture and induction, the expression level of the engineering bacteria is low (only 2mg/L of culture medium).

The present inventors found that the engineered bacteria grew slowly in the TG-20 medium containing high phosphorus, and cultured for 24 hours after inoculation, A₆₆₀Only 1-1.15, the expression level of rhEGF in low-phosphorus culture medium is low during induction, the expression level is about 2mg/L culture medium, and the product is mainly secreted into bacterial periplasm. Through testing various culture and induction conditions, the nutrient components in the culture medium are changed, and the engineering bacteria are cultured in the optimized Lpi culture medium for controlling the inorganic phosphorus content, so that the expression quantity is improved.

In the shake flask culture, in the low phosphorus Lpo culture medium, the rhEGF for inducing the expression can reach about 30mg/L culture medium.

In 15L fermentation tank culture, the rhEGF content in culture medium can be up to about 60mg/L culture medium, and in 150L fermentation tank culture, the rhEGF can be secreted and expressed by about 100mg/L culture medium or more.

Third, fermentation, separation and purification in pilot scale

1. Seed preparation:

plating glycerol engineering bacteria on a plate containing ampicillin (Ap), selecting a monoclonal, inoculating to an LB medium containing Ap, culturing at 37 ℃ overnight, inoculating to an enlarged culture the next day, and using as seeds for tank-loading fermentation.

2. Fermentation culture:

the seed liquid is inoculated into 100 liters of culture medium according to the proportion of 2 percent, fermented for about 18 hours at 37 ℃ and then placed in a tank.

3. Separating and purifying

High speed centrifugation, collecting supernatant of fermentation liquor, and ultrafiltration concentrating. Further, Phenyl Sepharose 6 Fastflow column, Q Sepharose Fast Flow column and Superdex 30 prep grade column were used for the chromatographic separation.

The flow of the pilot plant production process is shown in figure 8.

Pharmaceutical composition of human epidermal growth factor

The pharmaceutical composition for treating the peptic ulcer comprises the human epidermal growth factor or the derivative thereof and pharmaceutically acceptable auxiliary materials, wherein the content of the human epidermal growth factor or the derivative thereof is 0.001-2000 mu g/ml or 0.001-2000 mu g/g.

The pharmaceutical composition can be prepared into oral preparations and parenteral preparations.

The oral preparation comprises:

solid dosage forms, such as pills, granules, powders or discrete units such as tablets or capsules; liquid dosage forms in a form that is structured for ready use, such as a mixture, syrup, suspension or emulsion. The formulations may additionally contain diluents, powders, buffers, stabilizers, solubilizers, surfactants, preservatives, chelating agents and/or other pharmaceutical additives. Aqueous or non-aqueous vehicles or combinations thereof may be used, containing suitable sweetening, flavoring or similar substances as desired. For suspensions or emulsions, suitable thickeners, suspending agents or emulsifiers may be present. The pharmaceutical formulations may have slow, sustained or controlled release of the active ingredient provided by the matrix or diffusion control system.

Parenteral formulation:

for parenteral administration, rhEGF may be present in a sterile vehicle, either an aqueous or non-aqueous vehicle, or a combination thereof. Examples of excipients are water, ethyl oleate, derivatives of oils and polyols, glycols and their derivatives. It may contain additives commonly used in injectable formulations, such as stabilizers, solubilizers, pH modifiers, buffers, antioxidants, cosolvents, complexing agents, tonicity modifiers, etc. Some suitable additives are such as tartrate, citrate or similar buffers, alcohols, sodium chloride, dextrose and high molecular weight liquid polymers. Another form is sterile powder for reconstitution.

A preferred embodiment of the pharmaceutical composition of the present invention is an oral sustained release capsule. According to the result of EGF pharmacokinetic experiment, the oral EGF is stable in stomach, is degraded quickly after entering intestinal tracts and has low blood concentration. Therefore, the effect of oral EGF mainly occurs in the stomach. The longer EGF stays in thestomach, the better it promotes ulcer healing. The oral sustained-release capsule of the invention enters the stomach after oral administration, absorbs water to swell, forms hydrophilic gel, and slowly releases the medicine, and the release time lasts about 4 hours.

The invention applies the genetic engineering technology to successfully prepare the human Epidermal Growth Factor (EGF) from a laboratory to a pilot scale, finally realizes large-scale production, reduces the cost and provides quantitative guarantee for treating gastric ulcer by clinical application. The rhEGF preparation method of the invention adopts protein secretion expression, and has the following advantages:

(1) the expression product is free of Met (methionine) at its N-terminus and is identical to the sequence of a native protein in the organism.

(2) In E.coli, the secreted target protein has a better spatial folding conformation of the native protein, and thus has higher biological activity. The simple process is as follows: when a target protein peptide chain with a signal peptide is synthesized on a ribosome, the synthesized polypeptide chain is led to an inner membrane by a molecular chaperone such as SecB and the like, and is combined with a translocase consisting of factors such as SecA, SecY, SecE and the like on the membrane, a newly synthesized polypeptide molecule is translocated from the intracellular to the inner membrane by the translocase, and when passing through the inner membrane, the polypeptide chain is folded into a certain conformation, the signal peptide is cut off by a signal peptidase on the plasma membrane, and the factors SecD and SecF help the peptide chain to be dissociated from the inner membrane, and the disulfide bond isomerase on the plasma membrane enables the peptide chain to form a disulfide bond. The entire process allows the synthetic polypeptide chain of interest to fold into the conformation of the native protein and thus have high biological activity.

However, the in vivo expression product, either soluble, or inclusion body form, has one more Met at the N-terminus, which is different from the native protein sequence. The inclusion body formed by expression is a polypeptide random aggregation, has no biological activity, and must be subjected to a longer renaturation and refolding process to ensure that part of polypeptide chain has protein conformation. The level of biological activity depends on the efficiency of renaturation.

The preparation of the present invention adopts high molecular material with specific adhesion to mucomucin of digestive tract, such as carbomer (Carbopol), etc. to make medicine stay in specific position in digestive tract. And the rhEGF can be slowly and continuously released in the stomach, so that the medicine with higher concentration in the stomach is always kept, and the curative effect is better exerted.

FIG. 1 is the primary and secondary structure of hEGF.

Figure 2A is a synthetic hEGF sequence; FIG. 2B is the sequence of synthetic pho-P and sig.

FIG. 3A shows the construction and cloning of the synthetic hEGF gene; FIG. 3B shows the construction and cloning of synthetic pho-P and sig sequences. In the figure, E: EcoRI; h: HindIII; p: PstI.

FIG. 4 is a restriction map of pEG recombinant plasmid. In the figure, the plasmid is cut by EcoR I and HindIII; molecular weight labeling: pBR322+ Hinf I.

FIG. 5 is a schematic diagram of the construction of expression plasmid pAE-8.

FIG. 6 shows the determined nucleotide sequence of the synthetic hEGF target gene.

FIG. 7 shows the restriction enzyme map of constructed secretion expression plasmid pAE-8.

Fig. 8 is a pilot plant production process flow.

FIG. 9 is an electrophoretogram of periplasmic protein fractions of each EE-8 strain.

FIG. 10 shows an enzyme-cleaved diagram of a plasmid extracted from a passaging bacterium. Enzyme digestion: PstI + HindIII; molecular weight labeling: pBR322+ Hinf I.

FIG. 11 shows the relationship between the expression level of rhEGF and the induction time of engineering bacterium EE-8 in shake flask culture conditions, wherein inorganic phosphorus concentration (. mu.g/ml), □ bacterial A660 value, ○ hEGF content in periplasmic space (medium. mu.g/ml), △ hEGF content in medium (medium. mu.g/ml), and X hEGF content in cytoplasm (medium. mu.g/ml).

FIG. 12 shows rhEGF expression under fermentor culture conditions in which hEGF expression (mg/L) in the medium was expressed, □ glucose content (g/L), ○ inorganic phosphorus concentration (. mu.g/ml), △ bacterial A660 values.

FIG. 13 is a reduced SDS-PAGE gel scan.

FIG. 14 is a gel HPLC analysis.

FIG. 15 is an SDS-PAGE pattern.

FIG. 16 is an isoelectric focusing map.

Fig. 17 is an ultraviolet characteristic absorption spectrum.

FIG. 18 is a peptide profiling.

FIG. 19 is a graph showing the dose-effect relationship of rhEGF on gastric ulcer in rats treated with rhEGF for 12 days.

FIG. 20 is a graph of the effect of rhEGF on gastric ulcer in rats for 12 days.

FIG. 21 is a graph of ulcer index dose-effect relationship of pylorus ligation gastric ulcer model.

Fig. 22 is a graph of ulcer index of pylorus ligation gastric ulcer model.

Fig. 23 shows the percentage of ulcer occurrence in the pyloric ligation gastric ulcer model.

Fig. 24 shows the percentage of ulcer occurrence in the pyloric ligation gastric ulcer model.

FIG. 25 is a graph of dose-effect relationship for ethanol-damaged gastric ulcer models.

FIG. 26 is a graph of ulcer indices for an ethanol damaged gastric ulcer model.

The invention is illustrated below by means of examples and experimental examples, which are intended to illustrate the best mode of carrying out the invention. Various modifications made by persons skilled in the art in light of the teachings of this invention, which incorporate common general knowledge in the field, are within the scope of the claims appended hereto.

Example 1 screening of engineering bacteria

Each 20 transformed colonies were picked, inoculated into 3ml of high phosphorus medium (containing 50. mu.g/ml ampicillin) and grown overnight, and centrifuged the next day and then induced for 6 hours in a low phosphorus medium. 1ml of the bacterial suspension was centrifuged, and the obtained cell was suspended in 150. mu.l of a solution containing lysozyme (1mg/ml), 20% W/V sucrose, 30 mmol/L LTris. Cl (pH8.0), 1 mmol/L EDTA, and allowed to stand in ice bath for 15 minutes. The supernatant was the periplasmic protein fraction after centrifugation. Taking 15 microliter for electrophoretic analysis, wherein the electrophoretic separation gel is 15% polyacrylamide gel, and dyeing by a silver staining method after electrophoresis.

FIG. 9 is an electrophoretogram of periplasmic protein fractions of each EE-8 strain. As canbe seen from FIG. 9, the amount of hEGF secreted and expressed by the EE-86 strain was the highest. The strain is selected as a genetic engineering strain (referred to as EE-8).

EXAMPLE 2 expression plasmid stability experiment

A. Subculturing by inoculating solution culture medium

The strain was diluted 1000 times and inoculated in LB (Ap50, i.e., ampicillin 50. mu.g/ml medium) medium and cultured overnight at 37 ℃ corresponding to 10 cell divisions. The above operations were repeated 10 times a day to obtain 10 strains in total. Each strain was diluted by a certain fold, spread on an LB plate, and cultured overnight at 37 ℃ to obtain 10 plates in total. 100 single colonies are picked out from each plate and spotted on an LB (Ap50) plate, the plates are cultured overnight at 37 ℃, 10 plates are obtained in total, and the number of colonies growing on each LB (Ap50) plate is calculated, so that the corresponding plasmid preservation rate of each generation can be obtained.

TABLE 1 preservation rates of plasmids in cells dividing by different generations

Cell division generation number 102030405060708090100
	Preservation ratio (%) 10010010010010010010010099100 of recombinant plasmid

The results in Table 1 show that the recombinant plasmid pAE-8 was not lost after 100 generations of cell division.

B. Plate passage

The strain was inoculated in LB solution medium (Ap50) and cultured overnight at 37 ℃. Get 10³The cell fluid was spread on LB plates and cultured overnight at 37 ℃. 100 single colonies were picked from the plates and inoculated onto LB (Ap50) plates overnight at 37 ℃ and counted. Single colonies were then picked from LB (Ap50) plates. Subculturing by inoculating, e.g. byBefore ten passages, the preservation rate of the plasmid was calculated, and the results are shown in Table 2.

TABLE 2 preservation of plasmid in cells at different passage numbers of plate passage

Number of passages of analysis 12345678910
	Preservation ratio (%) 100100100100100100100100100100 of recombinant plasmid

DNAs were prepared by selecting one colony from 10-generation inoculated LB (Ap50) plates, and 1. mu.g of each DNA was digested with PstI and HindIII, followed by 1% agarose electrophoresis, and the results are shown in FIG. 10.

FIG. 10 is an agarose electrophoresis pattern of a plasmid extracted from a passaged bacterium digested with PstI + HindIII. Wherein columns 1 and 11 are molecular weight markers pBR322+ Hinf I, and columns 2 to 11 are electrophoretograms of plasmids extracted from passage bacteria of generations 1 to 10, respectively.

EXAMPLE 3 alkaline phosphatase Activity assay

When the normal colibacillus is lack of inorganic phosphorus in the surrounding environment, alkaline phosphatase can be expressed and secreted to the outer membrane, and phosphorus-containing compounds are hydrolyzed and inorganic phosphorus is taken up for the growth of bacteria. When the host bacteria YK537 express abnormal alkalinephosphatase, the kit is used for determining the alkaline phosphatase activity of the host bacteria so as to detect the phenotypic characteristics of the host bacteria.

(1) The principle is as follows:

the enzyme activity was determined by measuring the amount of p-nitrophenol at a wavelength of 405 nm.

Definition of enzyme activity units:

1mM p-nitrophenol is released per liter of sample measured in 1mM units per hour.

(2) Reagent:

alkaline phosphatase determination kit (Shanghai Lizhu Dongfeng technology Co., Ltd.)

P-nitrophenol (A.R.)

(3) The preparation of the thallus containing alkaline phosphatase component:

① the engineered bacteria were cultured in an ampicillin-containing induction medium at 37 ℃ and 200rpm for 20 hours in a shake flask.

② the control TG-1 or host strain was incubated with the induction medium without ampicillin in a shake flask at 37 ℃ and 200rpm for 20 hours.

③ taking 40mL of the above bacterial liquid, adding 1mL of hypertonic solution, oscillating the suspended bacteria, standing at room temperature for 10 minutes, centrifuging at 10,000rpm for 5 minutes, discarding the supernatant, adding 0.6mL of ice water to suspend the bacteria, carrying out ice bath for 10 minutes, centrifuging at 10,000rpm for 10 minutes, and obtaining the supernatant as periplasmic space protein.

(4) The determination method comprises the following steps:

① A3 mM p-nitrophenol standard was prepared with distilled water.

② the substrate solution was prepared according to the kit instructions.

③ mu.l sample to be tested, blank (distilled water) and p-nitrophenol standard solution are taken, 980 mu.l substrate solution is added, 10 mu.l 8N NaOH solution is added after water bath at 37 ℃ for 1 hour, and the absorbance value D is measured at 405nm₀Then 1 drop of concentrated hydrochloric acid is added, and after uniform mixing, the absorbance value D is measured at 405nm_cTaking D as D₀-D_cAnd (6) performing calculation.

④ formula:

enzyme Activity (mM) ═ D-D_{Blank space})/(D-D_{Standard of merit})×3

Measurement results

	Blank space	3mM standard	EE-8	TG-1
					OD600	/	/	6.46	5.36
D value	0.177	1.279	0.168	0.234
					Enzyme Activity (mM)	/	/	0	0.155
Enzyme Activity/OD600	/	/	0	0.0290

EXAMPLE 4 Shake flask culture

The cultivation was carried out in 250ml shake flasks containing 50ml of medium, both the cultivation and the induction being carried out at 37 ℃. The formula of the culture medium is as follows:

each 50ml of the liquid medium contained (final concentration of ampicillin sodium: 100. mu.g/ml):

0.5g Yeast extract 0.05g

1M Tris Cl (pH7.2) 2.5ml glucose 0.5g

MgSO₄7H₂O0.05 g saturated phenol Red 100. mu.l

Table 3 shows the rhEGF expression levels under various culture and induction conditions.

TABLE 3 expression level of engineering bacteria EE-86 under different culture and induction conditions

Culture conditions	Induction conditions	Total expression amount (mg/L medium)
			TG-20	TG-1	2
TG-20 20％ cassamino acid	TG-1	2-3
			LB	TG-1	4-5
Lpi	Lpo					30

rhEGF expression product was quantitatively determined using a radioimmunoassay kit (Amersham).

The secretion behavior of the expression product rhEGF of the engineering bacteria EE-86 (abbreviated as EE-8) is changed along with the improvement of the expression quantity. When expressed low, the expressed rhEGF is secreted mainly intracellularly into the periplasm of the bacterium. Since the periplasmic space of bacteria is small, when highly expressed, the expressed rhEGF is taken into the culture medium from the periplasm.

(2) Expression of engineering bacterium EE-8 in Lpi medium: in the experiment, EE-8 glycerol was inoculated into 50ml of LB-like Lpi medium and cultured overnight at 37 ℃. The timing of the start of inorganic phosphorus reduction was 0, and we called Lpo for the medium in which inorganic phosphorus was reduced. Sample 2ml every 2 hours, assay A₆₆₀Inorganic phosphorus content and hEGF content of each component in the cells. Extracting the components in the fungus body: the supernatant after centrifugation is used as a culture medium component, the thallus is suspended in 150 microliters of lysozyme (1mg/ml), 20% W/V sucrose, 30m mol/LTris.CL (pH8.0)1m mol/L EDTA solution, the mixture is placed for 15 minutes in ice bath, and the supernatant after centrifugation is used as a periplasmic protein component. Subsequently, the cells were suspended in 150. mu.l of 0.1mol/L Tris.Cl (pH 8.0). Freezing in dry ice, thawing at 37 deg.C, repeatedly thawing for 3 times to lyse bacteria, centrifuging, and collecting supernatant as soluble protein component in cytoplasm. By using¹²⁵An I-hEGF radioimmunoassay kit (Amersham export epidemic growth factor, human, reagent pack for RIA) quantitatively determines the rhEGF content in components such as culture medium, bacterial periplasm and cytoplasmic soluble protein.

The relationship between the expression amount of rhEGF of engineering bacterium EE-8 and the induction time under the culture and induction conditions is shown in FIG. 11.

The engineering bacteria can continue to grow in a low-phosphorus Lpo culture medium until the engineering bacteria grow to A₆₆₀The growth is stopped slowly when the phosphorus content in the culture medium is reduced to be lower, the engineering bacteria begin to induce and synthesize rhEGF, and the expression quantity is increased along with the increase of the induction time. After 10 hours of induction, the expression reaches a maximum value, which can last up to 24 hours. Mature rhEGF synthesized and processed in the bacteria after 4 hours of induction has begun to be secreted into periplasm and culture medium, and with the increase of expression amount, the intracellular synthesized rhEGF is transported to the periplasm through the periplasm' channelIn the medium, about 90% of rhEGF was intracellularly secreted into the medium after 10 hours. Intracellular and periplasmic rhEGF content was very low after 24 hours and the product was almost totally secreted into the culture medium.

EXAMPLE 5 fermenter culture

The fermentation was carried out in a B Braun 15L fermentor. The glycerol strain was inoculated into 50ml of LB (Ap50) medium and cultured overnight at 37 ℃. The bacterial solution was inoculated into a fermenter. The culture medium is based on LB, and 10g/L glucose is added. The culture was conducted with aeration, pH was controlled at 7.0, and the fermentation temperature was 37 ℃. After 9 hours of fermentation, the feed was started and 100ml of 1 l medium was added. Timed sample measurement A₆₆₀Determining phosphorus, and measuring the rhEGF content in the culture medium, wherein the measurement shows that the rhEGF content in the culture medium is the highest and can reach about 60mg/L of the culture medium after 15-18 hours of fermentation. The results are shown in FIG. 12.

Example 6 engineering bacteria expression stability test

The plate was passaged for 10 generations, and 10ml of LB (Ap50) medium was picked and cultured overnight at 37 ℃ the next day with the culture broth used as a glycerol strain. After being placed at-70 ℃ for 6 months, the strain is inoculated to a shake flask for culture and induced to express, and Table 4 shows that the expression quantity of the engineering bacteria after passage can still reach about 30ml/L culture medium (induced for 10 hours). This indicates that the engineering bacteria EE-8 has stable expression.

TABLE 4 expression stability of the subcultured engineered bacteria

(1) Expression condition of primary engineering bacteria:

induction time (hours)	Bacterium A660	Phosphate salts (μg/ml)	Expressed hEGF (μ g/ml)
							Culture medium	Periplasm	Cytoplasm	Total amount of
			0	0.90	20	0.00					0.00	0.00	0.00
2	2.44	10					0.00	0.00	0.00	0.00
			4	3.08	2	2.00					1.40	2.00	5.40
6	3.72	2					8.40	1.40	3.00	12.80
			8	3.85	3	15.00					0.90	10.30	26.20
10	4.49	3					26.70	0.56	3.20	30.46
			24	4.77	2	29.04					0.00	0.07	29.11

(2) The expression condition of the subculture engineering bacteria is as follows:

induction time (hours)	Bacterium A660	Phosphate salts (μg/ml)	Expressed hEGF (μ g/ml)
							Culture medium	Periplasm	Cytoplasm	Total amount of
			0	1.04	22	0.00					0.00	0.00	0.00
2	2.87	10					0.00	0.00	0.00	0.00
			4	3.55	2	0.00					0.15	2.00	2.15
6	3.79	2					6.00	0.30	3.30	9.60
			8	4.00	1	10.00					0.45	12.00	22.45
10	4.40	1					23.40	0.15	5.50	29.05
			24	4.56	1	26.30					0.00	0.70	27.00

Example 7 Pilot-Scale fermentation and isolation purification

1. Fermentation of

Recombinant engineering bacteria E.coli EE-8(pAE-8) secreting and expressing human epidermal growth factor (hEGF) are adopted as strains.

Seed culture medium: LB Medium

Fermentation medium:

culture medium proportioning meter

Composition of culture medium	Proportioning% (w/v)	Manufacturer of the product	Batch number
				Polypepton	2	Japanese pharmaceutical Co Ltd	S328B
Yeast extract
		1	OXOID	085-50687
Anhydrous magnesium sulfate					0.05	Shanghai reagent four factory	970703
	Anhydrous glucose	3	Shanghai Laoze fine chemical plant	980218
Na2HPO4·12H2O					0.06	Shanghai Xinhua plant	980202
	NaH2PO4·2H2O	0.04	Shanghai Xinhua plant	980202

Plating glycerol engineering bacteria on a plate containing ampicillin (Ap), selecting a monoclonal, inoculating to an LB medium containing Ap, culturing at 37 ℃ overnight, inoculating to an enlarged culture the next day, and using as seeds for tank-loading fermentation.

The seed liquid is inoculated into 100 liters of culture mediumaccording to the proportion of 2 percent, fermented for about 18 hours at 37 ℃ and then placed in a tank. The expression amount is about 100mg.

2. Separating and purifying

Centrifuging by a high-speed GQ142 continuous centrifuge, and collecting the supernatant of the fermentation liquor. The supernatant of the fermentation broth was concentrated by ultrafiltration using Millipore MW 5,000 ultrafiltration membranes (three stacked) on a Pellicon ultrafilter from Millipore.

Adding solid ammonium sulfate into the ultrafiltrate until the final concentration is 10% (mass percent), filtering, loading on Phenyl Sepharose 6 Fast Flow column, eluting with phosphate buffer solution, collecting rhEGF-containing components, diluting with Tris&Cl (pH7.6) buffer solution, loading on Q Sepharose Fast Flow column, eluting with NaCl gradient, collecting rhEGF-containing components, concentrating the liquid volume, loading on Superdex 30 prep grade column, and collecting rhEGF peak.

Example 8 pilot product identification

(1) Protein content determination (quantitative using HPLC gel chromatography or lowry method):

hplc quantification:

HPLC (high performance liquid chromatography): EGF standard sample from WATERS company: internal rhEGF standard

HPLC gel column: ohpakkb-803, WATERS Inc

Mobile phase: ph6.8, 10mM PB loading: 20 μ l

Detection wavelength: flow rate at 280 nm: 1ml/min

The method comprises the following steps:

a. standard EGF solutions were prepared at concentrations of 0.5. mu.g/. mu.l, 1. mu.g/. mu.l, 2. mu.g/. mu.l, 4. mu.g/. mu.l and 8. mu.g/. mu.l, in that order.

b. And (3) taking 20 mu l of standard EGF solutions with different concentrations to walk on an HPLC gel column to obtain the integral area of the EGF gel chromatographic peak, and drawing a standard curve according to the relation between the EGF protein concentration and the EGF gel chromatographic peak integral area.

c. And (3) taking a proper amount of EGF unknown concentration samples to dilute the EGF unknown concentration samples to a curve concentration range, taking 20 mu l of EGF unknown concentration samples to be loaded on a column, and obtaining the protein concentration of the EGF unknown samples from the integral area of chromatographic peaks corresponding to a standard curve.

Lowry method quantification: the samples were quantified with reference to the kit instructions.

(2) Purity analysis:

A. reduced SDS-PAGE gel Scan:

SDS-PAGE was performed using 15% gel, and the loading amount of rhEGF was not less than 5. mu.g. After the electrophoresis, the sample was silver-stained and developed, and the purity was analyzed by a GS-670 scanner from BIORAD. As a result, the purity of three rhEGF samples was greater than 95% (see FIG. 13).

B. Gel HPLC analysis:

the instrument comprises the following steps: waters600 high pressure liquid chromatograph

And (3) analyzing the column: TSK-GEL G3000 pw (7.5 I.D.. times 300mm)

Solution: 10mM pH6.8 phosphate buffer

Flow rate: 1ml/min

Detection wavelength: 280nm

Sample loading amount: 20 μ l

As a result, the purity of three rhEGF samples was greaterthan 95% (see FIG. 14).

(3) And (3) measuring the molecular weight:

SDS-PAGE was performed using 15% gel, and after completion of electrophoresis, silver staining was performed, and the internal rhEGF standard, which was calibrated by molecular weight, was used as a control. The three rhEGF samples were in perfect agreement with the rhEGF internal standard electrophoresis position (see FIG. 15).

(4) Isoelectric point determination:

the electrophoresis was performed on a BioRad Mini IEF Cell by isoelectric focusing. 5% polyacrylamide gel and amphoteric electrolyte with pH of 3-10 are used for electrophoresis at 100V for 15 minutes, 200V15 minutes and 450V1 hours, and Coomassie brilliant blue is used for staining after electrophoresis is finished. The results showed that the isoelectric point of the rhEGF sample was pH4.7 (see FIG. 16).

(5) Measurement of ultraviolet characteristic absorption spectrum:

the instrument is BECKMAN DU-600, double distilled water is used as blank, scanning is carried out within the wavelength range of 200-300 nm, and the wavelength and the absorption value of a characteristic absorption peak are recorded (see figure 17).

(6) Analysis of the N-terminal 15 amino acid sequence:

the rhEGF sample is analyzed on a PE-ABI 491A type sequence analyzer for an N-terminal amino acid sequence. The measured N-terminal sequence is:

ASN-SER-ASP-SER-GLU-CYS-PRO-LEU-SER-HIS-ASP-CLY-TYR-CYS-LEU。

(7) peptide profiling analysis:

and (3) enzymolysis conditions: the sample was dissolved in 100mM pH8.3NH₄HCO₃(1. mu.g/. mu.l), TPCK-trypsin (sample: enzyme: 20: 1w/w) was added, and HPLC analysis was performed after incubation at 37 ℃ overnight.

The instrument comprises the following steps: HP 1090 HPLC instrument

Operating temperature: 40 deg.C

And (3) analyzing the column: ABI RP-18 (30X 2.1mm I.D.)

Solution: 0.1% TFA (A) to 0.08% TFA/ACN (B)

Gradient: 0-45 min, 0-70% B

Detection wavelength: 220nm

Flow rate: 0.2ml/min

Sample loading amount: 10 μ l

The results showed that the rhEGF sample was completely digested with enzyme to show 4 peaks, and the results were identical between different batches (see FIG. 18).

(8) EGF content assay (immunological activity assay):

the content (immunological activity) of human epidermal growth factor is measured by ELISA kit of R&D SYSTEM company, and the specific operation method is shown in the kit specification.

And (4) calculating a result:

all absorbance values were subtracted from blank well absorbance values and used for calculation. Taking the logarithm value of the light absorption value (A) of the series of standard products as a vertical coordinate, taking the logarithm value of the EGF concentration (C) as a horizontal coordinate, and obtaining a standard curve after regression: log C ═ alog A + b

The EGF concentration of the sample is calculated according to the formula and then multiplied by the dilution factor to obtain the EGF concentration.

(9) And (3) biological activity determination:

the measurement was carried out by using a method of promoting Balb/c3T3 cell proliferation, and staining with MTT.

A. Preparation of test samples

The sterile glove is worn by hands, 3 capsules of each batch number are taken in an ultra-clean bench, the capsules are opened, the content and the capsule shells are put into a 500ml serum bottle (a sterile stirrer is put in advance) filled with 450ml of detection culture solution (RPMI 1640 culture solution added with 0.4% FBS from GIBCO company), the serum bottle is covered with a cover and then put into a magnetic stirrer to be stirred for 6 hours in an environment at 4 ℃, then the sample is taken out and centrifuged for 10 minutes at 12,000rpm, the supernatant is taken out, the supernatant is subjected to sterile filtration and is pre-diluted to 20ng/ml (200 mul of filtrate is taken and added into 800 mul of detection culture medium to be mixed evenly), and then the bioactivity is measured.

B. Cell culture and assay for biological Activity of test samples

a. Plate preparation: balb/c3T3 cells in logarithmic growth phase were collected, suspended in complete culture

In the solution (10% FBS was added to the RPMI 1640 culture solution from GIBCO Co., Ltd.), the cell concentration was adjusted to6.0×10⁴Perml, inoculated in a 96-well plate at 100. mu.l/well with 5% CO at 37 ℃₂The culture was carried out overnight.

b. Starvation: the supernatant was aspirated, and 100. mu.l/well of test medium (0.4% FBS in RPMI 1640 medium from GIBCO Co.) was added thereto at 37 ℃ with 5% CO₂The culture was carried out overnight.

c. Sample adding: diluting rhEGF sample and national reference substance with detection culture solution to a series of dilutions, removing supernatant from 96-well plate, adding diluted rhEGF sample 100 μ l/well, repeating the dilution steps for2 times, standing at 37 deg.C and 5% CO₂The culture box is filled for 72 hours.

d. Color development: adding 10. mu.l/well of MTT solution at 37 ℃ with 5% CO₂Culturing for 4 hours, sucking

80. mu.l/well of the supernatant was removed, and 100. mu.l/well of DMSO lysate was added thereto at room temperature

After standing for 30 minutes, the OD was measured₅₇₀The value is obtained.

e. And (4) processing a result: result meter on computer by SIGAPLOT analysis software

And calculating to obtain the half-effect dilution.

Biological activity (IU/ml) of sample is national reference biological activity

And (3) measuring results:

batch number 980401980702981003

Specific activity (1X 10)⁶IU/mg) 1.12 1.14 1.28

(10) Determination of residual DNA content:

the determination method comprises the following steps: DNA probe hybridization method

The kit comprises: the company Boehringer Dig High Prime DNA Labeling and DetectionStarter Kit I

Preparing engineering bacteria DNA: using phenol-chloroform extraction, see in particular methods in molecular cloning

Sample treatment: mu.g of the sample was added with 2. mu.l of 10 Xdigestion buffer (0.1M pH7.5Tris. Cl; 0.05M EDTA, 0.2% SDS), 1. mu.l of proteinase K (1mg/ml), mixed well, digested at 37 ℃ for 4 hours, placed in a boiling water bath for 5 minutes, and placed in an ice bath for further use.

And (3) hybridization membrane: Hybond-N from Amersham⁺Film

Pre-hybridization solution: 5 XSSC, 0.1% (w/v) N-Lauroylsarcosine, 0.02% SDS, 10% Blocking Reagent (supplied by kit). Experimental procedures were performed according to the method provided by Boehringer, Inc

The assay results showed that the residual DNA content in the 30. mu.g rhEGF sample was less than 50 pg.

(11) Determination of the content of residual ampicillin:

and (3) determining strains: ampicillin sensitive Staphylococcus aureus 9341a, supplied by the FDA of the United states

Culture medium: U.S. first, offered by Shi Gui Bao Co

The culture conditions are as follows: 16 to 18 hours at 37 DEG C

The results showed that there was no residual ampicillin in any of the three rhEGF samples.

(12) Pyrogen test (limulus reagent method):

the measurement was carried out by the method shown in the specification using a kit from Limulus tridentatus (Tachypleus tridentatus) Koch. The results show that the endotoxin content of the unit dose samples was less than 5 EU.

The results of pilot product identification are shown in table 5.

Example 9 prescription screening

Prescription screening was performed as per table 6.

TABLE 6

Group of	1	2	3	4	5
						rhEGF(μg)	15	15	15	15	15
Dextrin (mg)	70	65	63	60	55
						Hydroxypropyl methylcellulose (mg)	20	25	27	30	35
Carbomer (mg)	10	10	10	10	10
						Swelling time (hours)	3	4	4	5	5

Groups 2, 3, 4 and 5 with swelling time of 4-5 hours were selected for the release rate measurement. The results of the release test are shown in Table 7.

TABLE 7

Mean percent dissolution	2	3	4	5
					15min	46％	39％	29.4％	10％
30min	68％	46％	40.8％	16.5％
					60min	75％	60％	56％	28.5％
120min	90％	76％	71％	43％
					240min	103％	95％	90％	58.5％

According to the dissolution rate result of each component, the capsule preparation is prepared by selecting the group 4.

EXAMPLE 10 Capsule preparation 1 formulation prescription (1000 capsules)

Human recombinant epidermal growth factor (rhEGF) 15mg

Dextrin 60g

Hydroxypropyl methylcellulose 30g

Preparation process of carbomer 934P 10g2.

The dextrin, hydroxypropyl methylcellulose and carbopol were all sieved through 100 mesh sieve. Mixing the human recombinant epidermal growth factor with the above adjuvants, wet granulating with 80% ethanol solution, vacuum drying at 40 deg.C, sieving with 30 mesh sieve, measuring content, and making into capsule. Each capsule contains rhEGF15 μ g. 3. Sources and quality standards of auxiliary materials

Quality standard of raw and auxiliary materials sources recombinant human epidermal growth factor (hEGF) at Shanghai Biochemical institute of Chinese medicine and biological product institute

Quality Standard dextrin measurement of moisture, microorganism, Capsule content uniformity and Capsule Release level of the finished product of example 11, China pharmacopoeia 95 edition of hydroxypropyl methylcellulose DOW, Inc. of Shanghai glucose refinery, China pharmacopoeia 95 edition of Cambo (934P) of Goodrich corporation, USA, China pharmacopoeia 95 edition

(1) Water content: the method is shown in appendix VIII L of Chinese pharmacopoeia 95 edition. The results show that the weight loss of three batches of rhEGF preparations is less than 10.0%.

(2) Microorganism (mold, bacteria, escherichia coli) assay:

the method is shown in appendix XIJ of Chinese pharmacopoeia 95 edition. The results show that the three batches of rhEGF preparation all meet the requirements of pharmacopoeia.

(3) And (3) measuring the content uniformity of the capsules:

taking 10 rhEGF capsules, opening capsule shells, respectively placing the contents in volumetric flasks with the volume of 100ml, adding about 60ml of hydrochloric acid solution containing 0.1% bovine serum albumin (each 1000ml of double distilled water contains 1 g of bovine serum albumin and 9ml of concentrated hydrochloric acid), manually shaking for 1 hour at room temperature to completely swell the granules, standing for removing foams, fixing the volume by using the solution, and uniformly mixing. Taking 1ml to a 1.5ml centrifuge tube for each sample, centrifuging at 12,000rpm for 5 minutes, taking 20 mu l of supernatant, adding 980 mu l of diluent (1 XRD 5E) provided by a kit, mixing uniformly, taking 20 mu l, adding 780 mu l of diluent (1 XRD 5E) for two-stage dilution, wherein the total dilution multiple is 2000 times, and directly taking 200 mu l of the mixed solution as a sample for rhEGF content determination.

After the rhEGF standard curve is prepared, the rhEGF standard curve is calculated according to a log C ═ a log A + b equation and then multiplied byThe EGF concentration value of the sample can be obtained by diluting with a factor of 2000, and the result is XXX pg/ml, or XXX 10^-3ng/ml. The results are shown in Table 5.

(4) And (3) measuring the capsule release degree:

the measurement of the release rate is carried out according to the method in the appendix XD of the 95 th edition of Chinese pharmacopoeia.

The dissolution medium is a hydrochloric acid solution containing 0.1% bovine serum albumin (each 1000ml volume of double distilled water contains 1 g bovine serum albumin and 9ml concentrated hydrochloric acid), the volume is 500ml, and a basket rotating method is adopted. Taking 6 rhEGF capsules, measuring the temperature at 37 ℃, and rotating the rotary basket at 100 rpm. 0.5ml of the sample was sampled at 0.25, 1 and 4 hours, centrifuged at 12,000rpm for 5 minutes, 20. mu.l of the supernatant was taken, 280. mu.l of a diluent (1 XRD 5E) supplied from a kit was added, 20. mu.l of the mixture was mixed, 380. mu.l of the diluent (1 XRD 5E) was added thereto, and two-stage dilution was carried out, the total dilution ratio was 300-fold, and 200. mu.l of the mixture was mixed to prepare a sample directly and measured for rhEGF content.

The rhEGF concentration value of the test sample is calculated according to a standard curve equation and then multiplied by the dilution factor of 300, and the result is XXX pg/ml or XXX 10^-3ng/ml, divided by 30000pg/ml or 30ng/ml (concentration value after complete release of rhEGF in the capsule) is the value of the percentage released at the sampling time point. The results are shown in Table 5.

The results of the pilot product and the finished product are shown in Table 5.

TABLE 5 summary of pilot product and finished product identification results

Example 12 oral recombinant human epidermal growth factor (rhEGF) pharmacodynamic study

Experimental materials and methods

(I) test materials

Clean and closed groups of SD rats with the weight of 200-.

The tested drugs rhEGF, cimetidine and sucralfate are all prepared by 0.9% sodium chloride solutionThe dosage is respectively as follows: kg of rhEGF5, 10, 15, 30, 60ug^-1(ii) a Cimetidine 2mmol.kg^-1(ii) a Sucralfate 100mg. The control group was 1 ml/time of 0.9% NaCl.

(II) Experimental method

1. Model of gastric ulcer caused by absolute ethyl alcohol

Three days before the experiment, all the medicines are diluted by normal saline, after 48 hours of fasting, each mouse is perfused with 1ml of absolute ethyl alcohol, after 1 hour, the mouse is killed, the stomach is dissected and taken out, and 1 percent formaldehyde solution is fixed. Cutting and flattening along the lateral greater curvature of the stomach, observing the injury condition by naked eyes, measuring the injury length, expressing the injury degree by an ulcer index and performing the optical microscopy on pathological tissue sections. Injury index scoring criteria: linear lesions, scored 1 point per mm length, doubled for lesions with width over 2mm, summed up to give the index of ulceration of gastric mucosal lesions per rat, and compared between groups.

2. Water immersion stress type gastric ulcer model

Feeding the medicine three days before the experiment, after fasting for 24 hours, putting the rat into a special small iron cage, vertically immersing the rat into water with the temperature of 23 +/-0.5 ℃, immersing the rat to the level of the xiphoid process of the animal, taking the rat out after 24 hours, cutting off the neck to kill the rat, opening the abdominal cavity, ligating the cardia and the pylorus of the stomach, injecting 6ml of 1 percent formaldehyde into the gastric cavity, taking the stomach out, immersing the stomach into a formaldehyde solution, splitting the stomach along the greater curvature of the stomach after 30 minutes, counting the ulcer index appearing in the stomach of the gland, and performing comparison among groups according to the same measuring method.

3. Pylorus ligation type gastric ulcer model

Rats are dosed for 2 days, fasted for 48 hours, water is freely drunk, animals are anesthetized by ether, the abdominal cavity is opened to ligate pylorus, the abdominal cavity is opened again for 6 hours after operation to take gastric juice, the neck is cut off for death 18 hours after operation, the stomach is dissected and taken, 1% formaldehyde solution is injected into the gastric cavity, after 10 minutes, the stomach is cut along the greater curvature of the stomach, the area of the gastric ulcer before the gastric ulcer is counted, the sum is taken as an ulcer index, and then the ulcer inhibition percentage and the ulcer occurrence percentage are further calculated according to the ulcer index.

4. Acetic acid burning type gastric ulcer model

Before the experiment, the stomach was fasted for 24 hours, water was freely drunk, the abdominal cavity was opened under ether anesthesia, a glass tube with an inner diameter of 5mm and a length of 30mm was vertically placed on the serosal surface of the stomach, 0.2ml of glacial acetic acid was added into the tube cavity, glacial acetic acid was dipped with a cotton swab after 1.5 minutes, the incision of the operation was closed, normal diet was performed after the operation, a control group and an administration group were randomly set up the next day, the administration was continued for 12 days, the stomach was dissected and taken out, and fixed with formaldehyde, the ulcer area was measured, and the comparison was performed between the groups.

5. Aspirin (ASA) -induced gastric ulcer model

Dosing before experiment and fasting for 48 hours, freely drinking water, administrating 50mg of ASA (aspirin) per mouse per day of the experiment, 1 time per 1 hour,3 times continuously, killing after neck breaking at 4 hours, dissecting and taking out the stomach, cutting along the greater curvature of the stomach to flatten, observing the injury condition with naked eyes, counting the ulcer index of glandular stomach, and comparing among groups.

(III) measurement of gastric fluid amount, gastric acidity and pepsin

1. Measurement of gastric juice amount and gastric acidity:

gastric juice of rats 6 hours after pylorus ligation was collected, centrifuged at 1,000-2,000g for 5 minutes to remove gastric contents such as food debris, and the gastric juice was accurately weighed using a measuring cylinder of appropriate capacity. The acidity of the gastric juice was measured by an acidimeter.

2. Determination of free hydrochloric acid and total acidity:

reagent

(1)0.1mol/L sodium hydroxide solution;

(2) free hydrochloric acid indicator: 0.5% of p-dimethyl azoaniline acetic acid solution;

(3) total acid indicator: 0.5% phenolphthalein ethanol (95%) solution.

Method of producing a composite material

(1) Measuring gastric juice 5ml, placing in a triangular flask, adding equal amount of distilled water, and mixing well.

(2) Adding 2 drops of free acid indicator and total acid indicator, mixing with gastric juice, and getting cherry red if containing hydrochloric acid.

(3) 0.1mol/L NaOH was added dropwise to the flask via a burette, and the flask was shaken while adding the NaOH until the red color disappeared and orange color began to appear. The end point of the free acid is recorded as the number of ml of NaOH solution removed.

(4) Andcontinuously adding 0.1mol/L NaOH until the red color does not become dark any more. The end point of total acidity is recorded, and the total amount of NaOH solution used for the two titrations is recorded.

And (3) calculating:

total secretion amount ═ gastric juice amount × acidity 3. pepsin activity assay: capillary process using Mett

Unit of pepsin-average value²×16

(IV) statistical methods

The experimental data are counted by adopting a computer software package of SAS6.11 version, mean values are represented by +/-standard deviation, variance analysis and Q test are carried out, and the difference is represented by the p value being less than 0.05.

Second, experimental results

(ii) pathological changes

Ethanol and water immersion stress, pylorus ligation and an ASA model are not used, the gastric mucosa of a control group is obviously injured, the gastric mucosa is shown to have wide cord-shaped congestion, edema, erosion and bleeding on the mucosal surface, the gastric contents of part of specimens are blood-based but have no ulcer and perforation, the acute injury of the gastric mucosa is shown, and the reaction degree of the gastric mucositis of rats of the rhEGF group is reduced along with the increase of the dosage.

The gastric ulcer model burned by acetic acid is killed after being fed with the medicine for 12 days, slow ulcer which is consistent with the shape of the glass tube appears on the corresponding gastric mucosa surface burned by the acetic acid, and mucosa of other parts is complete. The posterior wall of the stomach specimen is adhered to the liver or intestinal wallat the ulcer forming position without perforation. The optical microscopy is shown in the following photographs. Obvious ulcer can be seen in a control group and a treatment group, the tissue structure of the gastric ulcer has a typical histological structure of the gastric ulcer, the tissue structure is shown as inflammatory exudate consisting of leucocytes and cellulose, necrotic tissue, granulation tissue layer and partial scar tissue, but the phenomena of thickening and narrowing of small vessel wall, adhesion between ulcer marginal mucosa muscle layer and the like are not seen, the change of peripheral chronic inflammation is not obvious, and the control group and EGFrug^-1The composition has more exudate and necrotic tissue, and has obvious inflammation, and the EGF amount is increased to treatThe treatment group had a greater tendency to heal, with less visible granulation and scar tissue, inflammatory exudate and necrotic tissue.

(II) gastric ulcer index

1. Acetic acid burning gastric ulcer model

60 rats were fasted for 24 hours and then subjected to the acetic acid burning gastric ulcer model, 5 were dead within 48 hours after surgery, and were all gastric perforations, which were not included in the study, and the remaining days were randomly given drugs in groups, and after 12 days, the gastric ulcer area was counted by laparotomy, and the results are shown in Table 8 and FIGS. 19-20. As shown in the figure, rhEGF10ug was therapeutically effective in the above groups compared to the control group in an amount-effect relationship. Compared with the cimetidine and sucralfate groups, the rhEGF10ug has no obvious difference, which indicates that the rhEGF has equivalent curative effect with the cimetidine and sucralfate.

TABLE 8 treatment of gastric ulcers in rats with rhEGF for 12 days ulcer index

Group N X + -S P

Control 726.00. + -. 10.17

Cimetidine 611.08 +/-8.44<0.05

Sucralfate 714.04 +/-6.65<0.05

rhEGF5ug 7. 18.21±7.51 ＞0.05

rhEGF10ug 7 13.89±4.79 ＜0.05

rhEGF15ug 7 11.71±5.45 ＜0.05

rhEGF30ug 7 9.43±5.06 ＜0.05

rhEFF60ug 7 7.64±3.61 ＜0.05

F ═ 5.25P ═ 0.0002 (significant difference between groups)

P value is compared between each treatment group and the control group

The EGF groups were not significantly different from the cimetidine and sucralfate groups.

2. Pylorus ligation gastric ulcer model

After being taken by 55 rats, after being fasted for 3 days for 48 hours, pylorus is ligated, after being operated for 6 hours, the abdominal cavity is opened again to extract gastric juice to determine gastric acidity and pepsin activity, after being molded for 18 hours, the gastric juice is taken to count ulcer indexes, and the results are shown in table 9 and figures 21-22, and compared with the control group, rhEGF5ug^-1The effect of the groups was not obvious, the ulcer index decreased with the increase of the dose in the groups of more than 10ug, the rhEGF group was compared with the cimetidine group, the other groups were not statistically different except for the difference of 5ug, and the ulcer inhibition percentage and the ulcer occurrence percentage ratio were shown in Table 9 and FIGS. 21-22.

TABLE 9 ulcer index of pyloric ligation gastric ulcer model

Group N X + -S P

Control group 78.57. + -. 3.79

Cimetidine 73.14 + -1.63<0.05

rhEGF5ug 8 8.13±4.67 ＞0.05

rhEGF10ug 8 5.63±2.50 ＞0.05

rhEGF15ug 8 2.75±2.31 ＜0.05

rhEGF30ug 8 1.75±1.75 ＜0.05

rhEGF60ug 91.13 + -1.36<0.05F ═ 7.29P ═ 0.0001 (difference between groups is significant) P is compared between each treatment group and the control groupThe EGF groups were not significantly different from the cimetidine group except for the 5ug group.

TABLE 10 percent inhibition of ulcer and percent ulcer development in pyloric ligation model

Percentage of inhibition of ulcer in group% percentage of ulcer occurrence%

Cimetidine 63.3685.71

rhEGF5ug 5.13 100

rhEGF10ug 34.36 100

rhEGF15ug 67.91 75

rhEGF30ug 79.58 62.5

rhEGF60ug 91.13 50

3. Model of gastric ulcer caused by absolute ethyl alcohol

48 rats are perfused with 1ml of absolute ethyl alcohol in the stomach after 3 days of medication, the rats are sacrificed after 1 hour, the stomach is dissected and taken out, the injuryof the gastric mucosa of the non-medication medicine is serious, and the stomach of 2 rats is filled with blood liquid. The ulcer index is shown in table 4. Ulcer indexes of the groups except rhEGF5ug were significantly decreased compared to the control group. Compared with the cimetidine group, the difference is not obvious, and the rhEGF has the same curative effect on the aspect of resisting the ethanol damage of the stomach as the cimetidine.

TABLE 11 ulcer index of ethanol-damaged gastric ulcer model

Group N X + -S P

Control 742.14. + -. 12.29

Cimetidine 626.50 + -8.17<0.05

rhEGF5ug 6 38.33±10.5 ＞0.05

rhEGF10ug 7 27.42±8.89 ＜0.05

rhEGF15ug 7 23.57±10.32 ＜0.05

rhEGF30ug 8 17.25±7.83 ＜0.05

rhEGF60ug 713.57 + -6.75<0.05F-8.47P-0.0001 (significant difference between groups) P value is that the difference between EGF and cimetidine in each treatment group and the control group is not significant (P>0.05)

4. Water immersion stress gastric ulcer model

After being applied for 2 days, 50 rats are placed in a special iron cage for a limited cold water stress test, and 6 rats die within 24 hours, which are not listed in the research. The ulcer indexes of each group are shown in table 12, the results are similar to the model, the rhEGF5ug group has no obvious curative effect (P is more than 0.05), the difference of the rest groups is obvious compared with a blank control group, and the difference is not obvious compared with the cimetidine group, which indicates that the protective effect of the rhEGF on the gastric mucosa against stress injury is equivalent to the cimetidine.

TABLE 12 ulcer index of water immersion stress gastric ulcer model

Group N X + -S P

Control 735.85. + -. 5.49

Cimetidine 621.67 +/-7.09<0.05

rhEGF5ug 6 32.33±6.62 ＞0.05

rhEGF10ug 6 28.83±6.43 ＞0.05

rhEGF15ug 6 24.83±9.81 ＜0.05

rhEGF30ug 6 18.67±5.99 ＜0.05

rhEGF60ug 712.28 + -7.99<0.05F-8.67P-0.0001 (significant difference between groups) P value is that the difference between EGF and cimetidine in the treatment group and the control group is not significant.

Model of ASA induced gastric ulcer

49 rats were fed and fasted for 48 hours before the ASA induced gastric mucosal injury model was performed. As shown in Table 13, the ulcer index of rhEGF10 ug/kg/body group was lower than that of the control group, and the effect was equivalent to that of the cimetidine group.

TABLE 13 ulcer index of ASA induced gastric ulcer model

Group N X + -S P

Control 77.71. + -. 2.56

Cimetidine 73.57 + -1.71<0.05

rhEGF5ug 7 5.86±1.96 ＞0.05

rhEGF10ug 7 5.00±1.82 ＜0.05

rhEGF15ug 74.14±1.34 ＜0.05

rhEGF30ug 7 4.00±1.41 ＜0.05

rhEGF60ug 7 3.42±1.51 ＜0.05

F＝3.27 P＜0.05

Thirdly, the gastric juice amount, gastric acidity and pepsin of rats

As shown in Table 14, the gastric juice amount was between 5.19 and 7.28, there was no significant difference between the groups, and the pepsin activity was between 4.07 and 4.39, and there was no statistical difference. Table 15 shows the pH of gastric juice in each group, which was significantly higher than that of the blank control group, but was not significantly different from that of the cimetidine group. There were significant differences between EGF5 and 30 ug/kg/group compared to the cimetidine group, but there were no statistical differences between EGF60 ug/kg/group. It is suggested that EGF in large dose has the effects of inhibiting gastric acid secretion and raising gastric juice pH. But the total acid and the free acid are not reduced remarkably.

TABLE 14 gastric juice volume and pepsin Activity in rats gastric pylorus ligated for 6 hours

Group N gastric juice volume (X + -Sml)^*Pepsin (X + -S)^**P

Control group 77.28 + -1.784.25 + -0.65

Cimetidine 86.20 + -2.334.58 + -1.08>0.05

EGF5ug 8 8.19±2.91 4.39±0.24 ＞0.05

EGF10ug 8 6.95±3.09 4.31±1.00 ＞0.05

EGF15ug 8 7.25±1.56 4.22±0.60 ＞0.05

EGF30ug 8 6.06±3.514.07±0.62 ＞0.05

EGF60ug 8 6.87±2.46 4.36±1.05 ＞0.05

*F＝0.56 P＝0.7591

**F＝0.30 P＝0.9332

TABLE 15 gastric juice pH of rats

Group N X + -S P1P 2

Control group 71.34. + -. 0.13

Cimetidine 81.89 + -0.56<0.05

EGF5ug 8 1.41±0.21 ＞0.05 ＜0.05

EGF10ug 8 1.30±0.09 ＞0.05 ＜0.05

EGF15ug 8 1.33±0.06 ＞0.05 ＜0.05

EGF30ug 8 1.48±0.28 ＞0.05 ＜0.05

EGF60ug 9 1.56±0.19 ＞0.05 ＞0.05

F＝4.80 P＝0.006

P1 comparison of treatment groups with placebo

P2 EGF groups compared to cimetidine group

TABLE 16 Total gastric acidity, free acid in rat

Group N Total acidity (X. + -. S)^*Free acid (X + -S)^**P1 P2

Control 74.89. + -. 0.873.06. + -. 0.29

Cimetidine 83.91 + -1.822.48 + -1.20>0.05

EGF5ug 8 4.22±1.04 3.05±0.82 ＞0.05 ＞0.05

EGF10ug 8 4.74±0.95 3.23±0.65 ＞0.05 ＞0.05

EGF15ug 8 4.73±0.41 3.10±0.24 ＞0.05 ＞0.05

EGF30ug 8 4.39±0.80 3.11±0.90 ＞0.05 ＞0.05

EGF60ug 9 4.00±0.71 2.98±0.42 ＞0.05 ＞0.05

*F＝1.09 P＝0.3817

**F＝0.81 P＝0.5654

P1 comparison of treatment groups with placebo

P2 EGF groups compared to cimetidine group

Example 13 toxicology Studies of oral recombinant human epidermal growth factor (rhEGF)

1. Acute toxicity test of oral recombinant human epidermal growth factor (hEGF) in rats

Healthy adult SD rats (supplied by BK) are 6-8 weeks old, 150-200 g in weight, and 20 rats in each half of male and female.

Rats were fasted for 6 hours and then orally administered 750. mu.g/kg body weight of rhEGF solution freshly prepared with physiological saline at a dose capacity of 1ml/100g body weight.

Feeding and observing for 14 days at the room temperature of 20 +/-1 ℃ and the relative humidity of 65-70%. Within 14 days, the activity, diet and weight gain of the rats are all normal. No other drug-related abnormalities were found.

Experiments show that the LD of rhEGF orally taken by rats₅₀More than 750 mug/kg body weight, which reaches 50 times of effective dose of rats.

2. Long-term toxicity test of oral recombinant human epidermal growth factor (hEGF) in rats

Rats 5-6 weeks old, 80-90 g in weight, half of male and female, are raised in cages, 5 rats per cage are raised at room temperature of 20-25 ℃, 50-70% in relative humidity, are fed by commercial pellet feed, and after one week of observation, 120 rats (half of female and male) which are healthy (for example, female must not be pregnant) are randomly selected and divided into 4 groups of low, medium and high doses and a control group. The administration components are respectively orally administered with recombinant human epidermal growth factor (rhEGF) at doses of 0, 30, 90 and 300 mug/kg/day (respectively equivalent to 30, 90 and 300 times of clinical dose); the control group was given an equal volume of distilled water. Rats were observed for toxicity and recovery 6 times a week for 26 weeks, with 8 weeks of withdrawal and dosing, and at 13 weeks, 26 weeks of withdrawal and 8 weeks of withdrawal animals were sacrificed 1/3 for hematological, hematological biochemical, urinary and pathological examinations.

The results showed that no other significant toxic effects were observed in relation to rhEGF, except for a slight decrease in body weight gain in female rats.

3. Long-term toxicity test of recombinant human epidermal growth factor on rhesus monkey

The rhesus monkey is fed with granulated feed (provided by Shanghai milk company) and an apple once every morning and afternoon, wherein the granulated feed and the apple are 100g each time. After adapting to the environment for 2 months and carrying out hematology, blood biochemistry, electrocardiogram and other examinations twice, 30 (15 female and male respectively) healthy animals are selected as the tested animals.

Oral administration long-term toxicity test was conducted using 24 rhesus monkeys, which were divided into 4 groups of 3 male and female groups, respectively, after layering by body weight. The oral preparation of the invention (containing rhEGF 64, 128 and 640 mug in each 100 mg) is adopted in 4 groups of low, medium and high doses and a control group, the dose of the administration group is respectively 16, 32 and 160mug/kg/day, the oral preparation containing only auxiliary materials and not containing rhEGF is taken orally in the control group, the oral preparation is taken before eating in the morning and 1 time per day, the administration is carried out for 26 weeks, and the recovery period after stopping taking the medicine is 8 weeks. General observation, hematology, blood biochemical index, urine examination, electrocardiogram, and fundus marrow examination are performed. 1/3 animals (1 in each case in female and male animals per dose group) were sacrificed at 13, 26 weeks of dosing and at the end of the drug withdrawal recovery period for pathological examination. The results showed that no toxic effects associated with rhEGF were seen in the above dose range.

Intravenous administration long-term toxicity test adopts 6 rhesus monkeys to randomly divide into 3 groups (rhEGF16, 32 and 160 mu g/kg/day) with low, medium and high doses, each group has 1 male and female, adopts the preparation of the invention to remove pyrogen freeze-dried product (each ampoule contains rhEGF 1mg), uses normal saline for injection to dilute to the required concentration, and injects 1ml/kg of intravenous injection once a day. One animal of each of the male and female animals was selected as a control group by oral administration and simultaneously injected with physiological saline. The administration period was also 26 weeks, and general observation, hematology, blood biochemical index, urine, electrocardiogram, eye ground and bone marrow examination were performed. The pathological examination was performed by killing 1 animal each at 13, 26 weeks of dosing and at the end of the recovery period from drug withdrawal. The results show that epithelial hyperplasia at the positions of bile duct, gastric gland, bladder and the like is observed in the animals of 32 and 160 mu g/kg/day dose groups after the administration for 26 weeks. The rest are normal.

3. Study of specific toxicology

(1) Teratogenicity test

In the teratogenic sensitive period (6-15 days of pregnancy), the SD pregnant rat is orally administrated with rhEGF at 30, 90 and 300ug/kg for ten consecutive days, and the test result shows that no embryo and embryo toxicity or abnormal fetus appears in each dose.

(2) Mutagenesis test (micronucleus test)

ICR mice are applied, the administration dosage is respectively 300ug/kg, 150ug/kg and 75ug/kg, the mice are killed by oral gavage and are taken for 24 hours, the femoral bone marrow is taken for measuring the micronucleus rate of cells, and the experimental result shows that the mice are negative.

(3) Tumorigenicity test

And observing the tumorigenicity of EGF by adopting a T cell immunodeficiency nude mouse in vivo inoculation method. Experiment, after inoculating human tumor HeLa cells, the EGF is infused at 80 mu g/kg/d and continuously po multiplied by 7d, and the EGF is observed to have no effect of stimulating the tumor growth after 3 weeks compared with a control group. EGF was then inoculated directly at 400. mu.g/kg (80. mu.g/0.2 ml/mouse) to the axilla of nude mice 3 and 12 weeks after subcutaneous injection, and necropsy was performed to visually observe autopsy and to check that no tumor was found at the injection site and in the lymph nodes, lung, kidney, spleen and liver. The result shows that EGF has no tumorigenicity to tumor-bearing nude mice or normal nude mice through the test.

Example 14 pharmacokinetic study of recombinant human epidermal growth factor (rhEGF)

1. Pharmacokinetics study of intravenous injection or oral recombinant human epidermal growth factor (rhEGF) in macaque

With R&ELISA kit produced byD system, the pharmacokinetics of the intravenous injection and oral administration of rhEGF of macaque are determined and studied. The methodological confirmation shows that the rhEGF is 0.76-781 pg at the concentration^-1The logarithm of the concentration in the range and the logarithm of the absorbance form good linear correlation; the lowest detection amount is 1.8pg.mL^-1(ii) a The internal precision is 0-8.8%; the batch precision is 2.9-24.2%; setting respective standard curves for each batch of tests, and calculating the concentration of an unknown sample; the recovery rate of the serum ranges from 88.5 to 105.9 percent, and the coefficient of variation of the recovery rate ranges from 2.2 to 11.7 percent. The methodology substantially meets the pharmacokinetic study requirements. Endogenous EGF in monkey serum is 29.8 +/-15.4 pg^-1And the normal value of the endogenous EGF in the human serum is within the range (0-622 pg.mL)^-1) Inner part^[1]. Orally administered 10, 40, 160 mug.kg^-1The rhEGF in the post-serum is obviously increased compared with the rhEGF in the pre-drug, but the increase value of the concentration, the peak concentration, the time to peak and the area under the curve (AUC) are not increased along with the increase of the administration dose, no statistical difference exists among dose groups, and the highest concentration of each monkey (32.4-375.9 pg mL)^-1) Still within the normal range of human values. The concentration decreased to the pre-drug level 6h after administration. Cross design comparison by itself 10 ug.kg per oral administration^-1The post bioavailability is 7.0 + -4.8%, and the dose is corrected and 40 μ g^-1Dose groups and 160. mu.g.kg^-1Mean biology of dose groupsThe utilization rates were 0.73% and 0.29%, respectively (P<0.05). Oral administration of rhEGF 40. mu.g.kg^-1.d^-1After x 7d, no increase or decrease of accumulation of blood concentration was observed. There was no statistical difference in plasma concentrations and pharmacokinetic parameters at the same time points after day 1 and day 7. The results show that rhEGF orally taken by macaques has obvious absorption, but a certain nonlinear saturation mechanism exists, so that the medicine absorption amount is limited, the individual difference of the absorption amount is very large, the absorption amount does not increase along with the increase of the dosage, the peak concentration reached after absorption still falls into the range of the normal endogenous EGF level of a human body, the level before administration is recovered after 6 hours, and the accumulation does not increase or decrease after continuous administration. The above characteristics suggest that there may be some kind of self-feedback regulation mechanism in vivo to maintain serum EGF at normal physiological requirement level, and avoid adverse effect of overhigh EGF level.

2. Research on absorption, metabolism, degradation, distribution and excretion of oral recombinant human epidermal growth factor (rhEGF) in gastrointestinal tract of animals

Selecting wistar rats, adopting oral administration and gastric lavage, respectively determining and researching by enzyme-linked immunosorbent assay, acid precipitation assay and radioactive tracer assay¹²⁵I-EGF and EGF destroy the rule of distribution, metabolism, excretion and absorption and degradation in digestive tract in the body of a rat.

(1) Chloramine T method isotope labeling preparation¹²⁵I-EGF, separated and purified by Sephadax G-75 and¹²⁵I-EGF radioactivity and chemical content measurement, obtaining the protein with specific activity of 1.90 mu ci/mu g¹²⁵I-EGF, reversed-phase high performance liquid chromatography, SDS-PAGE electrophoresis and TCA acid precipitation identification¹²⁵The radiochemical purity ofI-EGF is more than 96 percent,¹²⁵the results of the biological activity assay of I-EGF showed that the labeling with radioiodine was performed¹²⁵The I-EGF has better biological activity. Illustrating the preparation by the chloramine-T method¹²⁵I-EGF meets pharmacokinetic study requirements.

(2) Observation of gavage in rats¹²⁵Total radioactivity distribution of each tissue of the whole body at different times of 30min, 1h, 2h, 4h and 8h after I-EGF (40. mu.g/kg, 0.5 ml/rat) and settleable radioactivity distribution by TCA method, each time being 5 animals. The results show that the mouth isAdministration by oral administration¹²⁵I-EGF (40 mu g/kg), the total radioactive distribution capacity of all tissues of the whole body is greatly higher than the settleable radioactive distribution capacity of the TCA method, which shows that¹²⁵The great majority of I-EGF is metabolized and degraded by various proteases and digestive juice in the process of absorption in the digestive tract, and EGF prototypes are difficult to be directly absorbed into the body.¹²⁵The relative distribution capacity of I-EGF in tissue organs is highest in tissue organs such as stomach content, stomach wall, duodenum content, duodenum wall, jejunum content, jejunum wall, ileum content, ileum wall and the like, then thyroid, kidney, lung, liver, gallbladder, spleen, heart, adrenal gland, submandibular gland, large intestine wall, gonad, muscle, fat and the like, and the relative distribution of other tissue organs is relatively less, particularly in nervous tissues such as cerebellum, brain, spinal cord and the like, which indicates that the distribution capacity of I-EGF in tissue organs is relatively less, and the distribution capacity of I-EGF in nervous tissues and the like is relatively less, so that¹²⁵I-EGF may be more difficult to pass the normal blood-brain barrier.

(3) Observing EGF by adopting radioactive tracing TCA method and immunological method¹²⁵I-EGF (40. mu.g/kg) was administered orally and the absorption and degradation disruption of the EGF proto-drug or macromolecular fragment in rat stomach, jejunum, ileum at various times (3, 15, 30, 60, 120, 240 minutes) was measured in rat gastrointestinal tract or in tissue organs¹²⁵I-EGF or the prototype of EGF or the drug concentration of the immunologically active fraction. The results show that the rats are orally administered¹²⁵After I-EGF (40 mu g/kg), the EGF prototypes and the immunocompetent parts are kept at higher concentrations in the stomach, the gastric mucosa and the tissue of the gastric wall within 30 minutes, and the results measured by the radioactive tracing TCA method and the immunological method are basically consistent, which shows that the EGF is relatively stable and less damaged in the stomach within 30 minutes, and is measured by the radioactive tracing TCA method 2 hours and 4 hours after oral administration¹²⁵The concentration of I-EGF is still higher, but the concentration of the part with immunological activity of EGF measured by enzyme-linked immunosorbent assay is obviously reduced, and the result of HPLC method also proves that the EGF terminal segment is broken at the time, which indicates that the EGF prototype part is destroyed or inactivated under the action of gastric juice and various proteases.¹²⁵The distribution of I-EGF in tissues and organs such as jejunum, jejunum content, ileum content and the like is obviously lower than the prototypical EGF content of stomach content, gastric mucosa and stomach wall tissue at the same time, and the prototypical EGF content of ileum and ileum content isThe amount is obviously lower than the content of jejunum and jejunum, which shows that EGF is more stable in stomach and less degraded and damaged, and the most part of EGF is metabolized and degraded by various proteasesand digestive juice in the absorption process of small intestine, and the content of EGF prototypes at the lower section of digestive tract is lower.

(4) Oral administration for rats¹²⁵I-EGF (40 mu g/kg), the radioactive excretion rate of excrement, urine and bile at different time periods of 0-8h, 8-12h, 12-24h, 24-48h, 48-72h and the like and the cumulative total radioactive excretion. The results of the study showed that within 72 hours after administration, the rat feces and urine were present¹²⁵The total amount of radioactivity accumulated in the I-EGF accounts for 80.10% of the total amount of radioactivity administered orally, wherein the total amount of urine is about 64.20%, while the total amount of feces is only 15.89%, and most radioactivity in the urine is in the daily doseInternal drainage, i.e. cumulative volume drained within 24 hours, accounted for 56.20% of total dose of radiation administered orally.¹²⁵The radioactivity of I-EGF is excreted in the bile in a small amount, and the cumulative total excretion over 8 hours is about 2.05% of the total radioactivity administered orally. The results of TCA method determination show that prototypical EGF can not be detected in feces and urine, which indicates that EGF is mostly metabolized and decomposed by various proteases and digestive juice in the process of small intestine digestive tract absorption, and EGF entering into the body in a very small amount is also metabolized and destroyed by organs such as liver and the like. Therefore, the EGF orally taken is absorbed and metabolized to be excreted rapidly, and the main route of excretion is urine.

3. Metabolic degradation changes and absorption of recombinant epidermal growth factor (rhEGF) in situ ligated stomach or intestinal segments

By using¹²⁵I marker recombinant epidermal growth factor: (¹²⁵I-rhEGF) in combination with the reversed-phase high performance liquid chromatography (RHPLC) method, the metabolic degradation changes and absorption of rhEGF in rat in-situ ligated stomach or intestine were studied. The identification of RHPLC shows¹²⁵I-rhEGF was eluted at 44% acetonitrile with an elution purity of 96.3. + -. 0.4%. The biological activity of the marker rhEGF for stimulating Balb/c3T3 cell proliferation is similar to that of the polypeptide before the marker, EC₅₀29.7 and 31.5ng.mL, respectively^-1. Rat in situ ligation gastric infusion¹²⁵There was no significant change in total radioactivity of gastric contents 3h after I-rhEGF, but RHPCs analysis showed the appearance of marker-rhEGF radionuclides over timeBefore and after the peak appears, the shoulder appears bimodal or unchanged, which indicates that new components may appear at the position eluted by 40% acetonitrile, and in addition, trace hydrophilic property appears at the positions eluted by 17% and 0% acetonitrile¹²⁵I-labeling the degradent; the total radioactivity of the gastric system venous blood and the systemic venous blood is extremely low, and the RHPCs also detect a trace amount of labeled prototype drugs. In situ ligation intestinal infusion¹²⁵The total radioactivity of the intestinal contents began to decrease 5min after I-rhEGF and decreased to 20% after 6 hours, and the RHPLC analysis showed that: intestinal contents¹²⁵The I-rhEGF peak was smaller and the hydrophilic marker-degradant peaks eluted at 17% and 0% acetonitrile were relatively higher. Total radioactivity of intestinal contents¹²⁵I-rhEGF radioactivity concentration decreased exponentially, terminal t_1/21.3 +/-1.0 h and 1.8 +/-0.6 h respectively, the total radioactivity of mesenteric venous blood and systemic venous blood is increased, and the RHPLC analyzes that the blood is mainly water-soluble¹²⁵And I, marking the degradation product.

4. Animal pharmacokinetics study of recombinant human epidermal growth factor (rhEGF) for oral administration or burn

Adopting SD rat, artificially making pylorus ligation and deep and shallow II degree (large and small area) burn model, and orally or externally administering rhEGF-I¹²⁵15μg.kg^-1、30μg.kg^-1And I¹²⁵Thereafter, Cpm numbers were read every 15 ', 30', 1h, 2h, 3h, 4h, 5h, 6h blood draw, EGF was determined, 7h after which the rats were sacrificed and viscerated, weighed, ground, and Cpm numbers were read and compared to physiological gastrointestinal model data.

The results show that: physiological gastrointestinal oral administration of rhEGF-I¹²⁵And simple I¹²⁵In the group, the blood Cpm number is increased, and the visceral Cpm number is distributed from high to low and is ranked uniformly, taking the stomach as the main. The Cpm number in the blood of the pylorus ligation group is also increased, but is not as much as that of the physiological gastrointestinal model. The external rhEGF for deep and shallow II degree burns showed that shallow II degree absorption was higher than deep II degree, organ Cpm readings were ordered with skin first and stomach second, but the above experiment was performedIn the rat blood, the EGF value is not increased, which indicates that rhEGF is mainly applied to skin and mucous membrane surfaces in oral administration or external application, rarely absorbs into blood by using a prototype structure and acts on other organs, so that the rhEGF is safe to be taken orally clinically.

Claims (10)

1. The engineering strain EE-8 is preserved in the China general microbiological culture Collection center with the preservation number of 02787-1.

2. The preparation method of the recombinant human epidermal growth factor comprises the following steps:

constructing and cloning the synthesized hEGF gene by using codon preferred by escherichia coli;

constructing and cloning alkaline phosphatase start gene and signal peptide sequence gene fragment phoA-P + sig;

constructing an expression plasmid pAE-8;

transforming host bacteria with the expression plasmid pAE-8 and screening to obtain an engineering strain EE-8;

culturing engineering bacteria EE-8 in an Lpi culture medium with controlled inorganic phosphorus content, and inducing and expressing rhEGF.

3. The method of claim 2, wherein the preferred codons of E.coli are:

5’AATTCCGACT CTGAATGCCC GCTGTCTCAC

GACGGTTACT GCCTACACGA TGGTGTTTGC

ATGTATATCG AAGCTCTGGA CAAATACGCG

TGCAACTGTG TTGTTGGTTA CATCGGTGAA

CGTTGCCAGT ACCGTGACCT GAAATGGTGG

GAACTGCGT 3’

4. the method of claim 2 wherein the expression plasmid pAE-8 is constructed by the steps of: the hEGF gene fragment and the phoA-P + sig gene fragment are connected, then the hEGF gene fragment and the phoA-P + sig gene fragment are connected with pTZ18R plasmid which is cut by PstI and Hind III enzyme, JM103 is transformed, and an expression plasmid pAE-8 is screened out.

5. The method of claim 2, wherein the host bacterium is escherichia coli YK537 deficient in alkaline phosphatase, characterized by the genes: f^-leuB6 thi hsdR hsdM lacy rpsL20 galk2 ara-14xyl-5 mtl-1 supE44 endl^-phoA8 recA1。

6. The method of claim 2, wherein the formulation of the Lpi medium to control inorganic phosphorus content is as follows:

each 50ml of the liquid medium contained (final concentration of ampicillin sodium: 100. mu.g/ml):

0.5g Yeast extract 0.05g

1M Tris Cl (pH7.2) 2.5ml glucose 0.5g

MgSO₄7H₂O0.05 g saturated phenol Red 100. mu.l

7. The method of claim 2, further comprising a pilot scale fermentation and separation purification process, wherein the fermentation medium comprises Polypepton 2%, yeast extract 1%, anhydrous magnesium sulfate 0.05%, anhydrous glucose 3% and phosphate buffer, the seed solution of the engineering bacteria is expanded and inoculated into 100L fermentation medium at a ratio of 2%, fermented at 37 ℃ for 18 hours, and then subjected to high speed centrifugation, ultrafiltration concentration and chromatographic separation.

8. A pharmaceutical composition for treating peptic ulcer comprises human epidermal growth factor or its derivatives and pharmaceutically acceptable adjuvants, wherein the content of human epidermal growth factor or its derivatives is 0.001-2000 μ g/ml or 0.001-2000 μ g/g.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable excipients are dextrin, hydroxypropyl methylcellulose, and carbomer.

10. The pharmaceutical composition of claim 8, in the form of a capsule.

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