阅读说明：本技术 一种医用重组人源胶原蛋白及其制造方法 (Medical recombinant human collagen and preparation method thereof ) 是由 范小晴 于 2020-05-08 设计创作，主要内容包括：本发明提供一种医用重组人源胶原蛋白。所述医用重组人源胶原蛋白所述蛋白为单链结构,所述蛋白的等电点为6.8,所述蛋白包括氨基酸组数为268组。还提供一种医用重组人源胶原蛋白的制造方法,包括以下步骤：S1制备质粒；S2菌落转化；S3发酵培养；S4重组人源胶原蛋白的诱导；S5蛋白纯化,S1中的制备质粒：优选人员蛋白中的II型和III型胶原蛋白基因螺旋区的DNA片段。本发明提供的医用重组人源胶原蛋白具有可以充分的体现重组人源胶原蛋白使用时的水溶性更好的质量,并且可以适应使用者的体温标准,提高使用者使用重组人源胶原蛋白与人体的相容性,应用于人体上时减少人体的免疫排斥和过敏现象,方便对医药医疗的应用。(The invention provides a medical recombinant human collagen. The medical recombinant human collagen is of a single-chain structure, the isoelectric point of the protein is 6.8, and the number of the protein groups including amino acid groups is 268. Also provides a method for preparing the medical recombinant human collagen, which comprises the following steps: s1 preparing plasmids; s2 bacterial colony transformation; s3 fermentation culture; s4 induction of recombinant human collagen; purification of S5 protein, preparation of plasmid in S1: preferably DNA fragments of the helical region of the type II and type III collagen genes in human proteins. The medical recombinant human collagen provided by the invention can fully reflect the better water solubility quality of the recombinant human collagen when in use, can adapt to the body temperature standard of a user, improves the compatibility of the recombinant human collagen used by the user and the human body, reduces the immune rejection and allergy phenomena of the human body when applied to the human body, and is convenient for application to medical treatment.)

1. The medical recombinant human collagen is characterized in that the protein is of a single-chain structure, the isoelectric point of the protein is 6.8, and the number of the protein groups including amino acid groups is 268.

2. A method for preparing medical recombinant human collagen is characterized by comprising the following steps:

s1 preparing plasmids;

s2 bacterial colony transformation;

s3 fermentation culture;

s4 induction of recombinant human collagen;

and (4) purifying the S5 protein.

3. The method for producing recombinant human collagen for medical use according to claim 2, wherein the plasmid for production in S1:

preferably, DNA fragments of type II and type III collagen gene spiral regions in human protein are optimized, codon optimization and splicing recombination are carried out on the DNA fragments by utilizing a PCR technology, enzyme cutting sites of NcoI and Xho I added to primers are utilized, corresponding restriction enzymes are added to cut the gene fragments into sticky ends, the sticky ends are incubated with expression vectors of pET32a subjected to corresponding enzyme cutting treatment, T4DNA ligase is added, the sticky ends are connected for 12 hours at 4 ℃, and escherichia coli DH5 alpha is transformed; screening out white spot clone by using ampicillin sodium-LB plate, and extracting plasmid by using alkali cracking method or boiling method of molecular clone.

4. The method for producing recombinant human collagen for medical use according to claim 3, wherein the plasmid extraction is followed by the determination of the correct positive clone by digestion and PCR, and finally sequencing is performed to determine that the recombinant plasmid contains the correct gene sequence reading frame, and a successful recombinant expression vector named pET32a-NTOD is constructed.

5. The method for producing recombinant human collagen for medical use according to claim 4, wherein the colony transformation in S2 is:

screening single-spot clones from the screened correct pET32a-NTOD plasmid by using an ampicillin sodium-LB plate, culturing overnight in 5ml LB culture medium, transferring according to a ratio of 1:100, culturing in a shake flask at 36.5-37.2 ℃ until the isoelectric point is 0.5-0.7, adding ice-precooled sorbitol solution to mix the thalli evenly, coating the mixture on a culture substrate, and culturing for 2.5-3.5 days to obtain colonies.

6. The method for producing recombinant human-derived collagen for medical use according to claim 5, wherein the fermentation culture in S3 comprises:

transferring the cultured bacterial colony to YPD flat plates with the G418 concentrations of 0.5G/L, 1G/L, 2G/L, 3G/L, 4G/L and 5G/L by adopting a sterile transfer needle for culture, obtaining transformants after screening, inoculating the transformants obtained by screening into a culture medium, performing shake culture on the transformants in the culture medium for 24 hours, transferring the transformants after shake culture into a fermentation tank of the FBS culture medium, adjusting the pH value to be between 5.2 and 7, culturing for 18 to 21 hours, transferring the transformants after culture into a large tank of the FBS culture medium for fermentation, and controlling the growth temperature to be between 29.5 and 30 ℃.

7. The method for producing recombinant human-derived collagen for medical use according to claim 6, wherein the induction in S4 is:

the induction temperature is lower than the growth temperature, the pH is the same, the dissolved oxygen is controlled to be 22-28%, and the induction fermentation time is 38-44 hours.

8. The method for producing recombinant human collagen according to claim 7, wherein the protein conversion in S5 is:

and (3) resuspending the bacteria by using a Tris buffer solution, carrying out ultrasonic disruption, centrifuging, collecting supernatant, and purifying the supernatant by using a nickel ion affinity column to obtain the recombinant human collagen.

Technical Field

The invention relates to the technical field of medical protein, in particular to medical recombinant human collagen and a preparation method thereof.

Background

Collagen is a protein widely distributed in human connective tissues, is also the protein with the largest content in human bodies, and can account for 25-35% of the total amount of the protein. The collagen is a natural biological resource, has biological histocompatibility which is expected by other high polymer materials, and has supporting elasticity and degradability for cells, so the collagen can be widely applied to industries such as medicines, cosmetics and the like.

At present, the production of collagen mainly utilizes acid and alkali methods to treat tissues of animal sources and extract collagen from the tissues, but the obtained product has complex components and is mainly applied to feed addition and microbial fermentation culture media, Sichuan Ming allows biotechnology limited to adopt a bio-enzyme directional shearing technology, the produced collagen has stable composition and bioactivity, but the collagen products obtained by the methods have certain virus hidden dangers, and particularly easily cause xenogeneic rejection reaction when being applied to a human body, thereby limiting the application of the collagen in the aspect of medicine.

Through the recombination of the human collagen, when the recombined collagen is applied to the medical field, the affinity and the compatibility of the protein are poor, the temperature adaptation range of the protein is limited, and when the human body temperature is different, the recombined human collagen can not show the optimal effect.

Therefore, there is a need to provide a recombinant human collagen for medical use to solve the above technical problems.

Disclosure of Invention

The invention provides a medical recombinant human collagen, which solves the problem that the body temperature adaptation range of the recombinant human collagen is fixed.

In order to solve the technical problems, the medical recombinant human collagen provided by the invention has a single-chain structure, the isoelectric point of the protein is 6.8, and the number of the protein groups including amino acid groups is 268.

A method for preparing medical recombinant human collagen comprises the following steps:

s1 preparing plasmids;

s2 bacterial colony transformation;

s3 fermentation culture;

s4 induction of recombinant human collagen;

and (4) purifying the S5 protein.

Preferably, the plasmid prepared in S1:

preferably, DNA fragments of type II and type III collagen gene spiral regions in human protein are optimized, codon optimization and splicing recombination are carried out on the DNA fragments by utilizing a PCR technology, enzyme cutting sites of NcoI and XhoI added to primers are utilized, corresponding restriction enzymes are added to cut the gene fragments into sticky ends, the sticky ends are incubated with expression vectors of pET32a subjected to corresponding enzyme cutting treatment, T4DNA ligase is added, the sticky ends are connected for 12 hours at 4 ℃, and Escherichia coli DH5 alpha is transformed; screening out white spot clone by using ampicillin sodium-LB plate, and extracting plasmid by using alkali cracking method or boiling method of molecular clone.

Preferably, after plasmid extraction, positive clones are identified to be correct by enzyme digestion and PCR methods, and finally sequencing is performed to determine that the recombinant plasmid contains a correct gene sequence reading frame, so that a successful recombinant expression vector is constructed, and is named as pET32 a-NTOD.

Preferably, the colony transformation in S2:

screening single-spot clones from the screened correct pET32a-NTOD plasmid by using an ampicillin sodium-LB plate, culturing overnight in 5ml LB culture medium, transferring according to a ratio of 1:100, culturing in a shake flask at 36.5-37.2 ℃ until the isoelectric point is 0.5-0.7, adding ice-precooled sorbitol solution to mix the thalli evenly, coating the mixture on a culture substrate, and culturing for 2.5-3.5 days to obtain colonies.

Preferably, the fermentation culture in S3:

transferring the cultured bacterial colony to YPD flat plates with the G418 concentrations of 0.5G/L, 1G/L, 2G/L, 3G/L, 4G/L and 5G/L by adopting a sterile transfer needle for culture, obtaining transformants after screening, inoculating the transformants obtained by screening into a culture medium, performing shake culture on the transformants in the culture medium for 24 hours, transferring the transformants after shake culture into a fermentation tank of the FBS culture medium, adjusting the pH value to be between 5.2 and 7, culturing for 18 to 21 hours, transferring the transformants after culture into a large tank of the FBS culture medium for fermentation, and controlling the growth temperature to be between 29.5 and 30 ℃.

Preferably, the induction in S4:

the induction temperature is lower than the growth temperature, the pH is the same, the dissolved oxygen is controlled to be 22-28%, and the induction fermentation time is 38-44 hours.

Preferably, the protein conversion in S5:

and (3) resuspending the bacteria by using a Tris buffer solution, carrying out ultrasonic disruption, centrifuging, collecting supernatant, and purifying the supernatant by using a nickel ion affinity column to obtain the recombinant human collagen.

Compared with the related technology, the medical recombinant human collagen provided by the invention has the following beneficial effects:

the medical recombinant human collagen provided by the invention can fully reflect the better water solubility and quality of the recombinant human collagen in use, can adapt to the body temperature standard of a user, improves the compatibility of the recombinant human collagen used by the user and the human body, reduces the immune rejection and allergy of the human body when being applied to the human body, and is convenient for application to medical treatment.

Detailed Description

The present invention will be further described with reference to the following embodiments.

A medical recombinant human collagen comprises: the protein is of a single-chain structure, the isoelectric point of the protein is 6.8, and the number of the protein groups including amino acid groups is 268.

A method for preparing medical recombinant human collagen comprises the following steps: s1 preparing plasmids; s2 bacterial colony transformation; s3 fermentation culture; s4 induction of recombinant human collagen; and (4) purifying the S5 protein.

Preparation of plasmid in S1:

preferably, DNA fragments of type II and type III collagen gene spiral regions in human protein are optimized, codon optimization and splicing recombination are carried out on the DNA fragments by utilizing a PCR technology, enzyme cutting sites of NcoI and XhoI added to primers are utilized, corresponding restriction enzymes are added to cut the gene fragments into sticky ends, the sticky ends are incubated with expression vectors of pET32a subjected to corresponding enzyme cutting treatment, T4DNA ligase is added, the sticky ends are connected for 12 hours at 4 ℃, and Escherichia coli DH5 alpha is transformed; screening out white spot clone by using ampicillin sodium-LB plate, and extracting plasmid by using alkali cracking method or boiling method of molecular clone.

And after the plasmid extraction is finished, identifying the correct positive clone by using an enzyme digestion and PCR method, finally sequencing to determine that the recombinant plasmid contains a correct gene sequence reading frame, and constructing a successful recombinant expression vector named as pET32 a-NTOD.

Colony transformation in S2:

screening single-spot clones from the screened correct pET32a-NTOD plasmid by using an ampicillin sodium-LB plate, culturing overnight in 5ml LB culture medium, transferring according to a ratio of 1:100, culturing in a shake flask at 36.5-37.2 ℃ until the isoelectric point is 0.5-0.7, adding ice-precooled sorbitol solution to mix the thalli evenly, coating the mixture on a culture substrate, and culturing for 2.5-3.5 days to obtain colonies.

Fermentation culture in S3:

transferring the cultured bacterial colony to YPD flat plates with the G418 concentrations of 0.5G/L, 1G/L, 2G/L, 3G/L, 4G/L and 5G/L by adopting a sterile transfer needle for culture, obtaining transformants after screening, inoculating the transformants obtained by screening into a culture medium, performing shake culture on the transformants in the culture medium for 24 hours, transferring the transformants after shake culture into a fermentation tank of the FBS culture medium, adjusting the pH value to be between 5.2 and 7, culturing for 18 to 21 hours, transferring the transformants after culture into a large tank of the FBS culture medium for fermentation, and controlling the growth temperature to be between 29.5 and 30 ℃.

Induction in S4:

the induction temperature is lower than the growth temperature, the pH is the same, the dissolved oxygen is controlled to be 22-28%, and the induction fermentation time is 38-44 hours.

Protein conversion in S5:

and (3) resuspending the bacteria by using a Tris buffer solution, carrying out ultrasonic disruption, centrifuging, collecting supernatant, and purifying the supernatant by using a nickel ion affinity column to obtain the recombinant human collagen.

And (3) carrying out induced fermentation on bacterial colonies, carrying out resuspension on about 500ml of bacterial liquid precipitation by using a volume of 30-40ml, using lysozyme in combination with TritonX-100 to help to crack bacteria, carrying out ultrasonic bacteria breaking in an ice-water mixture environment, carrying out ultrasonic treatment every 2s, leaving 6s intervals and a total length of 45min, controlling the protection temperature at 40-46 ℃, carrying out centrifugation at 12200rpm/min for 20min, and collecting supernatant.

In this case, the solution contained a large amount of pET32a-NTOD recombinant protein.

Washing a nickel ion affinity column material by PBS buffer solution, mixing the column material with a solution of pET32a-NTOD for co-cultivation, gently shaking for 30min at room temperature or on ice, then loading the column, washing the hybrid protein by the PBS solution containing 15mM imidazole, leaving pure Trx-HC8 on the column, adding a proper amount of PPase protease with a His label on the column, gently shaking for 2 hours at room temperature or on ice, releasing the pET32a-NTOD protein from the column, eluting the pET32a-NTOD protein by the PBS solution, dialyzing the obtained product overnight, and freeze-drying the product into dry powder for later use.

Compared with the related technology, the medical recombinant human collagen provided by the invention has the following beneficial effects:

the quality of water solubility is better when the recombinant human collagen is used can be fully reflected, the body temperature standard of a user can be adapted, the compatibility of the recombinant human collagen used by the user and the human body is improved, the phenomena of immunological rejection and allergy of the human body are reduced when the recombinant human collagen is applied to the human body, and the application of the recombinant human collagen to medical treatment is facilitated.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present invention or directly or indirectly applied to other related technical fields are included in the scope of the present invention.