Recombinant expression plasmid, method for constructing recombinant humanized collagen escherichia coli, and preparation method and application of transdermal recombinant humanized collagen

Abstract

The invention discloses a recombinant expression plasmid, a method for constructing recombinant humanized collagen Escherichia coli and a preparation method and application of transdermal recombinant humanized collagen, and belongs to the technical field of polypeptide and genetic engineering. The recombinant humanized collagen Escherichia coli is constructed, and under induction of an inducer, the transdermal recombinant humanized collagen Escherichia coli is obtained. The bidirectional promoter initiates the polynucleotide of the recombinant III type human collagen and the polynucleotide of the recombinant III type human collagen to be expressed and split at the same time; and producing the recombinant II-type human collagen and the recombinant III-type human collagen. The method has the beneficial effects that the interference of homodromous expression is avoided; the recombinant II-type human collagen and the recombinant III-type human collagen are produced, and the recombinant II-type human collagen and the recombinant III-type human collagen have a synergistic effect, so that the collagen regeneration capacity and the transdermal effect are more excellent.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide and gene engineering, and more specifically relates to a recombinant expression plasmid, a method for constructing recombinant humanized collagen Escherichia coli, and a preparation method and use of transdermal recombinant humanized collagen. Background Art

[0002] Collagen is a type of protein widely distributed in human connective tissues. It is also the most abundant protein in the human body, accounting for 25% to 35% of the total protein. Its main functions are to maintain the extracellular environment, maintain the normal physiological functions of tissues and organs, and repair body damage. Collagen is a natural biological resource with bio-tissue compatibility, cell-supporting elasticity and degradability that other polymer materials cannot match. Therefore, collagen can be widely used in industries such as medicine and cosmetics.

[0003] The traditional method of producing collagen is to use acid, alkali, and enzymatic methods to treat animal tissues and extract collagen derivatives. The collagen extracted by these methods has lost its original biological activity and cannot be used in the biomedical field to play a real role. With the development of modern biotechnology, people continue to try to use transgenic technology to prepare recombinant human collagen in animal, plant and microbial expression systems, solving many shortcomings of traditional extraction processes. Foreign research institutions have obtained milk containing human collagen by breeding mice containing human collagen genes, but the cost of such production is too high and the production cycle is too long, so it cannot be put into large-scale production.

[0004] Chinese patent invention 201210482543.2 discloses a recombinant human collagen and a production method thereof, which discloses a recombinant human collagen obtained by connecting type III collagen peptide segments with a linker and adding a stabilizing sequence at the C-terminus.

[0005] Chinese patent invention 201811438582.6 discloses a polypeptide, a production method and use thereof, and utilizes genetic engineering technology to construct an engineered bacterium capable of preparing a polypeptide, and produces a polypeptide by the engineered bacterium, and finally prepares a polypeptide, namely, humanized collagen, by a step of enzymatic cleavage of the polypeptide by TEV protease; The present invention is a further improvement based on Chinese patent invention 201811438582.6, which discloses polypeptides, production methods and uses thereof. Since the invention requires the additional step of adding TEV protease to enzymatically cleave the polypeptide to finally prepare the polypeptide, i.e., humanized collagen, the conversion efficiency is low and the reaction cycle is long. Summary of the invention

[0006] In order to solve the above problems and overcome the shortcomings of the prior art, the present invention provides a polypeptide and a transdermal recombinant humanized collagen and uses thereof, which can effectively solve the problem of low conversion efficiency and long reaction cycle in the step of adding TEV protease to enzymatically cleave the polypeptide to finally prepare the polypeptide, i.e., humanized collagen, through engineering technology.

[0007] The specific technical solution of the present invention to solve the above technical problems is: a recombinant expression plasmid, the vector of the recombinant expression plasmid is pCOLADuet-1, and the recombinant expression plasmid comprises: two polynucleotides encoding genes of recombinant protein expression products and a bidirectional promoter for expressing and splitting the two encoding genes of recombinant protein expression products; The polynucleotide encoding the gene of the recombinant protein expression product includes a polynucleotide encoding a recombinant type II human collagen and a polynucleotide encoding a recombinant type III human collagen; The polynucleotide of the recombinant type III human collagen is a nucleotide sequence having n basic repeating units, the basic repeating unit is a nucleotide sequence as shown in SEQ NO: 1, n is an integer greater than or equal to 6, and each repeating sequence is directly connected; The polynucleotide of the recombinant type II human collagen is a nucleotide sequence having m basic repeating units, the basic repeating unit being a nucleotide sequence as shown in SEQ NO: 2; m is an integer of 2-3, and each repeating sequence is directly connected.

[0008] Furthermore, the nucleotide sequence of the bidirectional promoter is the nucleotide sequence shown in SEQ NO: 3; Furthermore, a method for constructing recombinant humanized collagen Escherichia coli using a recombinant expression plasmid; Step 1: Use primers 1 and 2 to forward connect the polynucleotide of recombinant type II human collagen to the vector pCOLADuet-1 to construct the expression vector pCOLA-iolM; Step 2: Using primers 3 and 4, the nucleotide sequence of the bidirectional promoter was connected to the expression vector pCOLA-iolM to construct the expression vector pCOLA-iolM-pLA; Step 3: Using primers 5 and 6, the polynucleotide of recombinant type III human collagen was reversely linked to the expression vector pCOLA-iolM-pLA to construct the expression vector pCOLA-iolM-pLA-iolG; Step 4: Introduce the expression vector pCOLA-iolM-pLA-iolG constructed in step 3 into the host cell Escherichia coli to obtain recombinant humanized collagen Escherichia coli.

[0009] The DNA sequence of primer 1 is: SEQ NO: 4; the nucleotide sequence shown is 3'-CGTCATATGAAAAACATCACCATCGGAATGG-5'; The DNA sequence of primer 2 is: SEQ NO: 5; the nucleotide sequence shown is 3'-GCTGGTACCTCATTAAGCAGATGGAACCAGCGCAC-5'; The DNA sequence of primer 3 is: SEQ NO: 6; the nucleotide sequence shown is 3'-GCTGGATCCAGGATGTCTCAGAGAAGTAAGTAC-5'; The DNA sequence of primer 4 is: SEQ NO: 7; the nucleotide sequence shown is 3'-GCAGTCGACTTAGGCTATTACATCGCGACGTTTTCC-5'.

[0010] The DNA sequence of primer 5 is: SEQ NO: 8; the nucleotide sequence shown is 3'-CGCCATGGTCATGACTCTTCGTATCGCCCTTTTCG-5'; The DNA sequence of primer 6 is: SEQ NO: 9; the nucleotide sequence shown is 3'-GCTGGATCCTTACTAAACGTTGGCAGGGTTGAGGG-5'.

[0011] A method for preparing transdermal recombinant humanized collagen, comprising culturing the recombinant humanized collagen Escherichia coli in a production medium, (1) Pick out the selected recombinant humanized collagen Escherichia coli colonies and culture them in LB medium at 37°C overnight; (2) The bacterial solution was inoculated and cultured at a ratio of 1:100, cultured at 37°C for 3 hours, and then the inducer was added for induction. The culture was continued at 20°C for 16 hours, and the bacteria were collected by centrifugation. (3) harvesting recombinant human type II collagen and recombinant human type III collagen; (4) Purifying the transdermal recombinant humanized collagen by Ni column and / or anion exchange chromatography.

[0012] Furthermore, the inducer is galactose.

[0013] Furthermore, the transdermal recombinant humanized collagen has transdermal properties.

[0014] Furthermore, the transdermal recombinant humanized collagen is used in cosmetics or health products. The beneficial effects of the present invention are: The present invention creatively prepares a recombinant expression plasmid and constructs a recombinant humanized collagen Escherichia coli. Under the induction of an inducer, a bidirectional promoter triggers the simultaneous expression and splitting of a polynucleotide of a recombinant type III human collagen and a polynucleotide of a recombinant type III human collagen, thereby avoiding the interference of co-directional expression; and produces recombinant type II human collagen and recombinant type III human collagen. The present invention creatively prepares transdermal recombinant humanized collagen, which contains recombinant type II humanized collagen and recombinant type III humanized collagen obtained by splitting, and has excellent transdermal effect; The present invention creatively prepares transdermal recombinant humanized collagen, which includes recombinant type II human collagen and recombinant type III human collagen obtained by splitting. The recombinant type II human collagen and the recombinant type III human collagen work synergistically to have a more superior collagen regeneration ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Attached Figure 1 is a comparison chart of the relative cell adhesion activities of the present invention and the comparative example; Attached Figure 2 It is a Sirius red staining picture; a is the Sirius red staining image of experimental group 1, i.e., recombinant type II human collagen; b is the Sirius red staining image of experimental group 2, i.e., recombinant type III human collagen; C is the Sirius red staining image of experimental group 3, i.e., recombinant type II human collagen and recombinant type III human collagen; d is the Sirius red staining image of experimental group 4, i.e. commercially purchased type III human collagen and commercially purchased type II human collagen; Attached Figure 3 The results of the Franz diffusion cell transdermal test of the transdermal recombinant humanized collagen prepared by the present invention; DETAILED DESCRIPTION

[0016] In the description of the present invention, specific details are only for a full understanding of the embodiments of the present invention, but those skilled in the art should know that the implementation of the present invention is not limited to these details. In addition, well-known structures and functions are not described or shown in detail to avoid blurring the key points of the embodiments of the present invention. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0017] Specific implementation of the present invention: In order to better understand the present invention, a specific embodiment is used for illustration. It is worth emphasizing that the effect of this embodiment is not substantially different from the various embodiments within the protection scope of the present invention, including the respective reagents and the content ratio of the reagents, and all of them can achieve the effects described in the present invention and solve the above-mentioned problems. Other combinations are not described here. A recombinant expression plasmid, wherein the vector of the recombinant expression plasmid is pCOLADuet-1, and the recombinant expression plasmid comprises: two polynucleotides encoding genes of recombinant protein expression products and a bidirectional promoter for expressing and splitting the two polynucleotides encoding genes of recombinant protein expression products; The polynucleotide encoding the gene of the recombinant protein expression product includes a polynucleotide encoding a recombinant type II human collagen and a polynucleotide encoding a recombinant type III human collagen; The polynucleotide of the recombinant type III human collagen is a nucleotide sequence having n basic repeating units, the basic repeating unit is a nucleotide sequence as shown in SEQ NO: 1, n is an integer greater than or equal to 6, and each repeating sequence is directly connected; The polynucleotide of the recombinant type II human collagen is a nucleotide sequence having m basic repeating units, the basic repeating unit being a nucleotide sequence as shown in SEQ NO: 2; m is an integer of 2-3, and each repeating sequence is directly connected.

[0018] The polynucleotide of the recombinant type III human collagen and the polynucleotide of the recombinant type II human collagen of the present invention are nucleotide sequences having multiple basic repeating units, provided that there is no linker between the repeating sequences. In this article, the linker can be more than one amino acid residue. The present invention has no limitation on the number of repeating sequences, as long as it can achieve the characteristics verified in the adhesion embodiment.

[0019] The polynucleotide sequences of the recombinant type III human collagen and the recombinant type II human collagen of the present invention should have an ENLYFQ sequence added to their N-termini during expression, which can be cut off by TEV protease to directly obtain the polynucleotides of the recombinant type III human collagen or the polynucleotides of the recombinant type II human collagen.

[0020] Furthermore, the nucleotide sequence of the bidirectional promoter is the nucleotide sequence shown in SEQ NO: 3; Furthermore, a method for constructing recombinant humanized collagen Escherichia coli using a recombinant expression plasmid; Step 1: Use primers 1 and 2 to forward connect the polynucleotide of recombinant type II human collagen to the vector pCOLADuet-1 to construct the expression vector pCOLA-iolM; Step 2: Using primers 3 and 4, the nucleotide sequence of the bidirectional promoter was connected to the expression vector pCOLA-iolM to construct the expression vector pCOLA-iolM-pLA; Step 3: Using primers 5 and 6, the polynucleotide of recombinant type III human collagen was reversely linked to the expression vector pCOLA-iolM-pLA to construct the expression vector pCOLA-iolM-pLA-iolG; Step 4: Introduce the expression vector pCOLA-iolM-pLA-iolG constructed in step 3 into the host cell Escherichia coli to obtain recombinant humanized collagen Escherichia coli.

[0021] The DNA sequence of primer 1 is: SEQ NO: 4; the nucleotide sequence shown is 3'-CGTCATATGAAAAACATCACCATCGGAATGG-5'; The DNA sequence of primer 2 is: SEQ NO: 5; the nucleotide sequence shown is 3'-GCTGGTACCTCATTAAGCAGATGGAACCAGCGCAC-5'; The DNA sequence of primer 3 is: SEQ NO: 6; the nucleotide sequence shown is 3'-GCTGGATCCAGGATGTCTCAGAGAAGTAAGTAC-5'; The DNA sequence of primer 4 is: SEQ NO: 7; the nucleotide sequence shown is 3'-GCAGTCGACTTAGGCTATTACATCGCGACGTTTTCC-5'.

[0022] The DNA sequence of primer 5 is: SEQ NO: 8; the nucleotide sequence shown is 3'-CGCCATGGTCATGACTCTTCGTATCGCCCTTTTCG-5'; The DNA sequence of primer 6 is: SEQ NO: 9; the nucleotide sequence shown is 3'-GCTGGATCCTTACTAAACGTTGGCAGGGTTGAGGG-5'.

[0023] A method for preparing transdermal recombinant humanized collagen, comprising culturing the recombinant humanized collagen Escherichia coli in a production medium, (1) Pick out the selected recombinant humanized collagen Escherichia coli colonies and culture them in LB medium at 37°C overnight; (2) The bacterial solution was inoculated and cultured at a ratio of 1:100, cultured at 37°C for 3 hours, and then the inducer was added for induction. The culture was continued at 20°C for 16 hours, and the bacteria were collected by centrifugation. (3) harvesting recombinant human type II collagen and recombinant human type III collagen; (4) Purifying the transdermal recombinant humanized collagen by Ni column and / or anion exchange chromatography.

[0024] Furthermore, the inducer is galactose.

[0025] Furthermore, the transdermal recombinant humanized collagen has transdermal properties.

[0026] Furthermore, the transdermal recombinant humanized collagen is used in cosmetics or health products. Embodiment 1: (1) Construction of recombinant expression plasmid and construction of recombinant humanized collagen Escherichia coli: Step 1: The pCOLADuet-1 plasmid was purchased as a vector, and the polynucleotide of the recombinant type II human collagen was forward-linked to the vector pCOLADuet-1 using primers 1 and 2 to construct the expression vector pCOLA-iolM; The polynucleotide of the recombinant type II human collagen is a nucleotide sequence having m basic repeating units, the basic repeating unit being such as SEQ NO: 2; m is an integer of 3, and each repeating sequence is directly connected.

[0027] Step 2: Using primers 3 and 4, the nucleotide sequence of the bidirectional promoter was connected to the expression vector pCOLA-iolM to construct the expression vector pCOLA-iolM-pLA; Wherein, the nucleotide sequence of the bidirectional promoter is the nucleotide sequence shown in SEQ NO: 3; Step 3: Using primers 5 and 6, the polynucleotide of recombinant type III human collagen was reversely linked to the expression vector pCOLA-iolM-pLA to construct the expression vector pCOLA-iolM-pLA-iolG; The polynucleotide of the recombinant type III human collagen is a nucleotide sequence having n basic repeating units, the basic repeating unit is a nucleotide sequence as shown in SEQ NO: 1, n is an integer greater than or equal to 6, specifically n is 6, and each repeating sequence is directly connected; Step 4: Introduce the expression vector pCOLA-iolM-pLA-iolG constructed in step 3 into the host cell Escherichia coli to obtain recombinant humanized collagen Escherichia coli.

[0028] (2) Preparation method of transdermal recombinant humanized collagen Step 5: Pick out the selected recombinant humanized collagen Escherichia coli single colony and culture it in LB medium at 35°C overnight; Step 6: Inoculate the bacterial solution and amplify the culture, culture at 35°C for 2 hours, add the inducer galactose for induction, continue to culture at 20°C for 15 hours, and collect the bacteria by centrifugation; Step 7: Harvesting recombinant human type II collagen and recombinant human type III collagen; Step 8: Purify the transdermal recombinant humanized collagen by Ni column and / or anion exchange chromatography.

[0029] Embodiment 2: (1) Construction of recombinant expression plasmid and construction of recombinant humanized collagen Escherichia coli: Step 1: The pCOLADuet-1 plasmid was purchased as a vector, and the polynucleotide of the recombinant type II human collagen was forward-linked to the vector pCOLADuet-1 using primers 1 and 2 to construct the expression vector pCOLA-iolM; The polynucleotide of the recombinant human type II collagen is a nucleotide sequence having m basic repeating units, the basic repeating unit being such as SEQ NO: 2; m is an integer of 2, and each repeating sequence is directly connected.

[0030] Step 2: Using primers 3 and 4, the nucleotide sequence of the bidirectional promoter was connected to the expression vector pCOLA-iolM to construct the expression vector pCOLA-iolM-pLA; Wherein, the nucleotide sequence of the bidirectional promoter is the nucleotide sequence shown in SEQ NO: 3; Step 3: Using primers 5 and 6, the polynucleotide of recombinant type III human collagen was reversely linked to the expression vector pCOLA-iolM-pLA to construct the expression vector pCOLA-iolM-pLA-iolG; The polynucleotide of the recombinant type III human collagen is a nucleotide sequence having n basic repeating units, the basic repeating unit is a nucleotide sequence as shown in SEQ NO: 1, n is an integer greater than or equal to 6, specifically 6, and each repeating sequence is directly connected; Step 4: Introduce the expression vector pCOLA-iolM-pLA-iolG constructed in step 3 into the host cell Escherichia coli to obtain recombinant humanized collagen Escherichia coli.

[0031] (2) Preparation method of transdermal recombinant humanized collagen Step 5: Pick out the selected recombinant humanized collagen Escherichia coli single colony, place it in LB medium, and culture it at 36°C overnight; Step 6: Inoculate the bacterial solution and amplify the culture, culture at 36°C for 3 hours, add the inducer galactose for induction, continue to culture at 22°C for 20 hours, and collect the bacteria by centrifugation; Step 7: Harvesting recombinant human type II collagen and recombinant human type III collagen; Step 8: Purify the transdermal recombinant humanized collagen by Ni column and / or anion exchange chromatography.

[0032] Embodiment 3: (1) Construction of recombinant expression plasmid and construction of recombinant humanized collagen Escherichia coli: Step 1: The pCOLADuet-1 plasmid was purchased as a vector, and the polynucleotide of the recombinant type II human collagen was forward-linked to the vector pCOLADuet-1 using primers 1 and 2 to construct the expression vector pCOLA-iolM; The polynucleotide of the recombinant type II human collagen is a nucleotide sequence having m basic repeating units, the basic repeating unit being such as SEQ NO: 2; m is an integer of 3, and each repeating sequence is directly connected.

[0033] Step 2: Using primers 3 and 4, the nucleotide sequence of the bidirectional promoter was connected to the expression vector pCOLA-iolM to construct the expression vector pCOLA-iolM-pLA; Wherein, the nucleotide sequence of the bidirectional promoter is the nucleotide sequence shown in SEQ NO: 3; Step 3: Using primers 5 and 6, the polynucleotide of recombinant type III human collagen was reversely linked to the expression vector pCOLA-iolM-pLA to construct the expression vector pCOLA-iolM-pLA-iolG; The polynucleotide of the recombinant type III human collagen is a nucleotide sequence having n basic repeating units, the basic repeating unit is a nucleotide sequence as shown in SEQ NO: 1, n is an integer greater than or equal to 6, specifically 7, and each repeating sequence is directly connected; Step 4: Introduce the expression vector pCOLA-iolM-pLA-iolG constructed in step 3 into the host cell Escherichia coli to obtain recombinant humanized collagen Escherichia coli.

[0034] (2) Preparation method of transdermal recombinant humanized collagen Step 5: Pick out the selected recombinant humanized collagen Escherichia coli single colony and culture it in LB medium at 35°C overnight; Step 6: Inoculate the bacterial solution and amplify the culture, culture at 35°C for 2 hours, add the inducer galactose for induction, continue to culture at 20°C for 15 hours, and collect the bacteria by centrifugation; Step 7: Harvesting recombinant human type II collagen and recombinant human type III collagen; Step 8: Purify the transdermal recombinant humanized collagen by Ni column and / or anion exchange chromatography.

[0035] Embodiment 4: (1) Construction of recombinant expression plasmid and construction of recombinant humanized collagen Escherichia coli: Step 1: The pCOLADuet-1 plasmid was purchased as a vector, and the polynucleotide of the recombinant type II human collagen was forward-linked to the vector pCOLADuet-1 using primers 1 and 2 to construct the expression vector pCOLA-iolM; The polynucleotide of the recombinant human type II collagen is a nucleotide sequence having m basic repeating units, the basic repeating unit being such as SEQ NO: 2; m is an integer of 2, and each repeating sequence is directly connected.

[0036] Step 2: Using primers 3 and 4, the nucleotide sequence of the bidirectional promoter was connected to the expression vector pCOLA-iolM to construct the expression vector pCOLA-iolM-pLA; Wherein, the nucleotide sequence of the bidirectional promoter is the nucleotide sequence shown in SEQ NO: 3; Step 3: Using primers 5 and 6, the polynucleotide of recombinant type III human collagen was reversely linked to the expression vector pCOLA-iolM-pLA to construct the expression vector pCOLA-iolM-pLA-iolG; The polynucleotide of the recombinant type III human collagen is a nucleotide sequence having n basic repeating units, the basic repeating unit is a nucleotide sequence as shown in SEQ NO: 1, n is an integer greater than or equal to 6, specifically 8, and each repeating sequence is directly connected; Step 4: Introduce the expression vector pCOLA-iolM-pLA-iolG constructed in step 3 into the host cell Escherichia coli to obtain recombinant humanized collagen Escherichia coli.

[0037] (2) Preparation method of transdermal recombinant humanized collagen Step 5: Pick out the selected recombinant humanized collagen Escherichia coli single colony, place it in LB medium, and culture it at 36°C overnight; Step 6: Inoculate the bacterial solution and amplify the culture, culture at 36°C for 3 hours, add the inducer galactose for induction, continue to culture at 22°C for 20 hours, and collect the bacteria by centrifugation; Step 7: Harvesting recombinant human type II collagen and recombinant human type III collagen; Step 8: Purify the transdermal recombinant humanized collagen by Ni column and / or anion exchange chromatography.

[0038] Embodiment 5: Same as embodiment 1, except that: The polynucleotide of the recombinant type III human collagen is a nucleotide sequence having n basic repeating units, the basic repeating unit is a nucleotide sequence as shown in SEQ NO: 1, n is an integer greater than or equal to 6, specifically 9, and each repeating sequence is directly connected; Embodiment 6: Same as embodiment 1, except that: The polynucleotide of the recombinant type III human collagen is a nucleotide sequence having n basic repeating units, wherein the basic repeating unit is a nucleotide sequence as shown in SEQ NO: 1, wherein n is an integer greater than or equal to 6, specifically 10, and each repeating sequence is directly connected.

[0039] In order to more intuitively demonstrate the process advantages of the present invention, the method of the present invention is compared with the method of equivalent replacement of the same process. Comparative Example 1: As a control experiment of the present invention, the recombinant type II human collagen and the recombinant type III human collagen obtained by the present invention were replaced with commercially available type II human collagen and type III human collagen by adding equal amounts; Comparative Example 2: By citing, the recombinant human collagen prepared by the national patent 201210482543.2 "A recombinant human collagen and its production method" was introduced; Comparative Example 3: As a control experiment of the present invention, the recombinant type II human collagen and recombinant type III human collagen obtained by the present invention are added in equal amounts to replace and add commercially available type I human collagen and type III human collagen in equal amounts; Detection methods and detection indicators 1. Collagen activity detection method 1. Use the UV absorption method to detect the concentration of the protein samples to be tested, including the control human collagen (Sigma), recombinant type II human collagen and recombinant type III human collagen samples. Specifically, the UV absorption of the samples at 215nm and 225nm is measured respectively. The empirical formula C (μg / mL) = 144X (A215-A225) is used to calculate the protein concentration. Note that the test must be performed when A215 < 1.5. The principle of this method is to measure the characteristic absorption of peptide bonds under far-ultraviolet light. It is not affected by the chromophore content, has few interfering substances, and is easy to operate. It is suitable for detecting human collagen and its analogs that do not develop color with Coomassie Brilliant Blue.

[0040] 2. Add 100 μl of various protein solutions and blank PBS solution as a control to the 96-well plate and let stand at room temperature for 60 min.

[0041] 3. Add 105 3T3 cells in good culture status to each well and incubate at 37 degrees for 60 minutes.

[0042] 4. Wash each well 4 times with PBS.

[0043] 5. Use LDH detection kit (Roche, 04744926001) to detect the absorbance of OD492nm. According to the value of blank control, the degree of cell adhesion can be calculated. The cell adhesion rate can reflect the activity of collagen. The higher the activity of protein, the better the external environment can be provided to cells in a short time, helping cells to adhere to the wall. Taking the adhesion rate of human collagen as 1, the relative cell adhesion activity of the collagen to be tested can be calculated. See for details. Figure 1 ; Depend on Figure 1 It can be seen that: The transdermal recombinant humanized collagen prepared by the present invention has a relatively excellent relative cell adhesion activity compared with comparative examples 1-3; wherein, (1) Compared with Comparative Example 1, The difference is that the present invention obtains recombinant type II human collagen and recombinant type III human collagen by gene construction, while comparative example 1 replaces commercially available type II human collagen and type III human collagen by adding; the relative cell adhesion activity of comparative example 1 is lower; It can be seen that the relative cell adhesion activities of recombinant human type III collagen and recombinant human type II collagen are different from those of commercially available human type II collagen and human type III collagen.

[0044] (2) Compared with Comparative Example 2, the present invention Although both the present invention and comparative example 2 use gene recombination to obtain recombinant humanized collagen, the recombination methods are different, and the nucleotide sequences of the recombinant type III human collagen are different, resulting in a lower relative cell adhesion activity of comparative example 2, even lower than that of comparative example 2. This may be because during co-directional transcription, the transcription of the upstream promoter will pass through the downstream promoter, which will affect the transcription of the downstream promoter to a certain extent and reduce the downstream transcription level.

[0045] The present invention creatively adopts a bidirectional promoter, forwardly connects the polynucleotide of recombinant type II human collagen to the nucleotide sequence of the bidirectional promoter, reversely connects the polynucleotide of recombinant type III human collagen, expresses and splits two segments of the bidirectional promoter encoding the recombinant protein expression product, and induces the bidirectional promoter with an inducer to trigger the simultaneous expression and splitting of the polynucleotide of recombinant type III human collagen and the polynucleotide encoding the recombinant type III human collagen; and produces recombinant type II human collagen and recombinant type III human collagen; avoiding the defects in comparative example 2.

[0046] (3) Compared with Comparative Example 3, The difference is that the present invention obtains recombinant type II human collagen and recombinant type III human collagen by gene construction, while comparative example 3 adds commercially available type I human collagen and type III human collagen of the same quality as recombinant type II human collagen and recombinant type III human collagen by addition; The cell adhesion activity of comparative example 3 is lower than that of the present invention, and is also lower than the relative cell adhesion activity of comparative examples 1 and 2; this indicates that different human collagens have a certain influence on the cell adhesion activity, and the synergistic effect of recombinant type II human collagen and recombinant type III human collagen is superior to type II human collagen and type III human collagen, and is better than type I human collagen and type III human collagen; This may be because human type I collagen is in the form of thick fibers in bundles, while recombinant human type II collagen and recombinant human type III collagen are in the form of reticular thin fibers. As components of the extracellular matrix, recombinant human type II collagen and recombinant human type III collagen support different cells to achieve various functions, especially transdermal absorption, promoting fibroblast metabolism and promoting collagen expression. As a further study of the present invention, a method for constructing recombinant humanized collagen Escherichia coli; Step 1: Use primers 1 and 2 to forward connect the polynucleotide of recombinant type II human collagen to the vector pCOLADuet-1 to construct the expression vector pCOLA-iolM; Step 2: Using primers 3 and 4, the nucleotide sequence of the bidirectional promoter was connected to the expression vector pCOLA-iolM to construct the expression vector pCOLA-iolM-pLA; Step 3: Using primers 5 and 6, the polynucleotide of recombinant type III human collagen was reversely linked to the expression vector pCOLA-iolM-pLA to construct the expression vector pCOLA-iolM-pLA-iolG; Step 4: Introduce the expression vector pCOLA-iolM-pLA-iolG constructed in step 3 into the host cell Escherichia coli to obtain recombinant humanized collagen Escherichia coli.

[0047] This study also further studied the effects of different primers on the construction of recombinant humanized collagen Escherichia coli of the present invention, so as to complete the induction of the bidirectional promoter by the inducer, trigger the simultaneous expression and splitting of the polynucleotide of recombinant type III human collagen and the polynucleotide encoding recombinant type III human collagen; and produce recombinant type II human collagen and recombinant type III human collagen; The study found that replacing the above primers would lead to a decrease in cell adhesion activity, especially after multiple passages, the bacteria showed a cliff-like decrease in cell adhesion activity. It is inferred that this may be due to the insufficient stability of the Escherichia coli constructed for recombinant humanized collagen, resulting in the loss of two polynucleotides encoding the recombinant protein expression product genes or the termination of transcription.

[0048] 2. Study on collagen regeneration after integration of transdermal recombinant humanized collagen with extracellular matrix (ECM) Separating the recombinant human type II collagen and the recombinant human type III collagen of the present invention, wherein the separation method includes but is not limited to affinity chromatography, ion exchange chromatography, gel filtration chromatography and immunological binding techniques, and any technique capable of separating the recombinant human type II collagen and the recombinant human type III collagen is within the scope of the present invention; The regeneration of collagen after integration with the extracellular matrix (ECM) was investigated and compared between different humanized collagens and the extracellular matrix (ECM). The working principle is: Sirius red staining can distinguish type I collagen (red) and type III collagen (green), and recombinant type II human collagen and recombinant type III human collagen are not stained due to differences in molecular weight.

[0049] Table 1: Comparative test results of Sirius red staining of different human collagens

[0050] in:

[0051] (Experimental group 1 is the recombinant type II human collagen group): 8 weeks after the recombinant type II human collagen was integrated with the extracellular matrix (ECM), there was no promotion of the regeneration of type I and type III collagen in the tissue. Figure 2 in, a; (Experimental group 2 is the recombinant type III human collagen group): 8 weeks after the recombinant type III human collagen was integrated with the extracellular matrix (ECM), it was observed that it significantly promoted the regeneration of type I and type III collagen in the tissue. For details, see the attached Figure 2 middle, b; (Experimental group 3 is the recombinant type II human collagen and recombinant type III human collagen group): 8 weeks after integration with the extracellular matrix (ECM), it can be observed that the regeneration of type I and type III collagen is obviously higher than that of the recombinant type III human collagen group in experimental group 2; see the attached Figure 2 in, c; (Experimental Group 4): Commercially available type III human collagen and commercially available type II human collagen were added. Sirius red staining showed a small amount of color development, which did not increase over time. See the attached Figure 2 in, d; From the above analysis, we can know that: The recombinant type II human collagen and recombinant type III human collagen groups have superior collagen regeneration capabilities; while recombinant type II human collagen, commercially available type III human collagen, and commercially available type II human collagen have no promoting effect on the regeneration of type I and type III collagen in tissues.

[0052] It can be seen from experimental groups 1 and 3 that although the recombinant type II human collagen group has no effect on promoting the regeneration of type I and type III collagen in tissues, it can promote the regeneration of type I and type III collagen in tissues that have not been promoted. The present invention further studies and finds that: although recombinant human type II collagen cannot promote the regeneration of type I and type III collagen in tissues, it can greatly promote the regeneration of type IV and type VII collagen, and type IV and type VII collagen are related to the repair of the skin basement membrane. Therefore, the repair of the skin basement membrane provides conditions for the regeneration of type I and type III collagen, and indirectly promotes the regeneration of type I and type III collagen. Recombinant human type II collagen and recombinant human type III collagen synergistically have a more superior collagen regeneration ability.

[0053] 3. Study on the transdermal effect of transdermal recombinant humanized collagen Franz diffusion cell transdermal test was used; Depend on Figure 3It can be seen that: the blue is the DAPI fluorescence of the cell nucleus, and the green is the fluorescence of the sample labeled with FITC. The transdermal recombinant humanized collagen prepared by the present invention has an excellent transdermal effect. Compared with the blank group, i.e. the control group without adding transdermal recombinant humanized collagen, the transdermal effect is 94% in 12 hours, and there is no significant difference between parallel experiments.

[0054] In summary, the present invention creatively prepares a recombinant expression plasmid and constructs a recombinant humanized collagen Escherichia coli. Under the induction of an inducer, a bidirectional promoter triggers the simultaneous expression and splitting of a polynucleotide of a recombinant type III human collagen and a polynucleotide of a recombinant type III human collagen; and produces recombinant type II human collagen and recombinant type III human collagen; The present invention creatively prepares transdermal recombinant humanized collagen, which contains recombinant type II humanized collagen and recombinant type III humanized collagen obtained by splitting, and has excellent transdermal effect; The present invention creatively prepares transdermal recombinant humanized collagen, which includes recombinant type II human collagen and recombinant type III human collagen obtained by splitting. The recombinant type II human collagen and the recombinant type III human collagen work synergistically to have a more superior collagen regeneration ability.

Claims

1. A method for preparing transdermal recombinant humanized collagen, characterized in that: The recombinant humanized collagen Escherichia coli is cultured in the production medium, including (1) Pick a single colony of recombinant humanized collagen Escherichia coli and culture it in LB medium at 35-38°C overnight; (2) Inoculate the bacterial solution and amplify the culture at 35-38°C for 2-4 hours, add the inducer for induction, continue to culture at 20-24°C for 15-20 hours, and collect the bacteria by centrifugation; (3) harvesting recombinant human type II collagen and recombinant human type III collagen; (4) Purifying the transdermal recombinant humanized collagen by Ni column and / or anion exchange chromatography.

2. The method for preparing transdermal recombinant humanized collagen according to claim 1, characterized in that The inducer is galactose.

3. The method for preparing transdermal recombinant humanized collagen according to claim 1, characterized in that The transdermal recombinant humanized collagen has transdermal performance.

4. The method for preparing transdermal recombinant humanized collagen according to claim 1, characterized in that The transdermal recombinant humanized collagen is used in cosmetics or health products.

5. A recombinant expression plasmid, wherein the vector of the recombinant expression plasmid is pCOLADuet-1, characterized in that Used to construct the recombinant humanized collagen Escherichia coli according to any one of claims 1 to 4, The recombinant expression plasmid comprises: two segments of polynucleotides encoding genes for recombinant protein expression products and a bidirectional promoter for expressing and splitting the two segments encoding genes for recombinant protein expression products; The polynucleotide encoding the gene of the recombinant protein expression product includes a polynucleotide encoding a recombinant type II human collagen and a polynucleotide encoding a recombinant type III human collagen; The polynucleotide of the recombinant type III human collagen is a nucleotide sequence having n basic repeating units, the basic repeating unit is a nucleotide sequence as shown in SEQ NO: 1, n is an integer greater than or equal to 6, and each repeating sequence is directly connected; The polynucleotide of the recombinant type II human collagen is a nucleotide sequence having m basic repeating units, the basic repeating unit being such as SEQ NO: 2; m is an integer of 2-3, and each repeating sequence is directly connected.

6. The recombinant expression plasmid according to claim 5, characterized in that The nucleotide sequence of the bidirectional promoter is the nucleotide sequence shown in SEQ NO:

3.

7. A method for constructing recombinant humanized collagen Escherichia coli; characterized in that: Utilizing the recombinant expression plasmid of claim 5, Step 1: Use primers 1 and 2 to forward connect the polynucleotide of recombinant type II human collagen to the vector pCOLADuet-1 to construct the expression vector pCOLA-iolM; Step 2: Using primers 3 and 4, the nucleotide sequence of the bidirectional promoter was connected to the expression vector pCOLA-iolM to construct the expression vector pCOLA-iolM-pLA; Step 3: Using primers 5 and 6, the polynucleotide of recombinant type III human collagen was reversely linked to the expression vector pCOLA-iolM-pLA to construct the expression vector pCOLA-iolM-pLA-iolG; Step 4: Introduce the expression vector pCOLA-iolM-pLA-iolG constructed in step 3 into the host cell Escherichia coli to obtain recombinant humanized collagen Escherichia coli.

8. The method for constructing recombinant humanized collagen Escherichia coli according to claim 7, characterized in that Said The DNA sequence of primer 1 is: SEQ NO: 4; The DNA sequence of primer 2 is: SEQ NO: 5; The DNA sequence of primer 3 is: SEQ NO: 6; The DNA sequence of primer 4 is: SEQ NO: 7; The DNA sequence of primer 5 is: SEQ NO: 8; The DNA sequence of primer 6 is: SEQ NO:9.

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