A preparation method for 293F cells expressing type III recombinant human collagen

Abstract

The present invention discloses a preparation method of 293F cells expressing type III recombinant human collagen, and relates to the field of biotechnology. The preparation method comprises the steps of obtaining the 293F cells expressing type III recombinant human collagen by fermentation and purification using recombinant engineering cells; the construction method of the recombinant engineering cells comprises the steps of transfecting a lentivirus carrying a P4HA gene and a P4HB gene and a lentivirus carrying a COL3A1 gene into a host cell to construct the recombinant cell line; the host cell is a 293F cell. The preparation method can realize the preparation of recombinant human collagen with a triple helix structure, self-assembly in vitro, and the same amino acid sequence as natural human collagen, and has important industrial application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a method for preparing type III recombinant human collagen expressed by 293F cells. Background Art

[0002] Collagen is the main component of the extracellular matrix and the most abundant and widely distributed protein in animals. It has a unique tissue structure and biological function, and plays an important role in maintaining the normal physiological functions and injury repair of cells, tissues, and organs.

[0003] At present, collagen can be divided into two categories according to its source, namely tissue-extracted collagen and bio-fermented collagen. Tissue-extracted collagen is collagen molecules extracted from various animal tissues, but its application is greatly limited due to its exogenous nature and risks such as other pathogenic microorganisms. Bio-fermented collagen is collagen obtained through engineered cell fermentation, also known as recombinant collagen.

[0004] According to the "Guidelines for the Naming of Recombinant Collagen Biomaterials" issued by relevant departments, recombinant type III collagen refers to "recombinant collagen based on the collagen COL3A1 gene or partial gene encoding", and can be distinguished according to the following aspects: ① Recombinant human collagen, the full-length amino acid sequence encoded by the human collagen specific type gene prepared by DNA recombinant technology, has a triple helix structure; ② Recombinant human collagen, the full-length or partial amino acid sequence fragment encoded by the human collagen specific type gene prepared by DNA recombinant technology, or a combination of functional fragments of human collagen; ③ Recombinant collagen-like protein, the amino acid sequence or fragment encoded by a specific gene designed and modified by DNA recombinant technology, or a combination of such functional amino acid sequence fragments. This gene coding sequence or amino acid sequence has low homology with the gene coding sequence or amino acid sequence of human collagen.

[0005] At present, the mainstream products on the market are recombinant human collagen or recombinant collagen-like proteins, because these collagens have no post-translational modification and triple helix structure, and can be expressed in large quantities in microorganisms at a low cost. However, these two collagens do not have a triple helix structure, and the internal enzyme cleavage sites are extremely accessible, so they are prone to enzymatic hydrolysis. In addition, due to the lack of steric hindrance, the flexible single chain is also prone to chemical reactions due to the lack of amino acid side chains, resulting in non-enzymatic hydrolysis. In addition, the mechanical properties of single-chain collagen are far inferior to those of triple helix collagen, and it is difficult to be competent in application scenarios that require higher mechanical strength. The expression level of recombinant human collagen is much lower than that of the other two collagens. Escherichia coli, yeast, plants, baculovirus, and mammalian cell expression systems have all been used to express recombinant human collagen. Although many expression systems are described in the literature, the current expression systems used for large-scale production of human collagen rely on yeast and plants.

[0006] Defects and shortcomings of existing industrial production technology: (1) The natural post-translational modification of yeast is different from that of mammalian cells, which may cause the recombinant protein produced to be different in structure and function from that produced in mammalian cells, affecting its effect in some applications that require strict protein modification. (2) The expression level of collagen expressed by plant cells is limited, and extraction and purification are difficult. Based on this, the present invention intends to express type III recombinant human collagen through 293F cells, reduce the immune rejection reaction of the recombinant protein, increase the expression level and simplify the purification process. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing type III recombinant human collagen expressed by 293F cells to solve the problems existing in the above-mentioned prior art. The preparation method can realize the preparation of recombinant human collagen with a triple helical structure, self-assembly in vitro, and the same amino acid sequence as natural human collagen.

[0008] To achieve the above object, the present invention provides the following solutions:

[0009] The present invention provides a method for constructing a recombinant engineered cell for expressing triple helical type III recombinant human collagen, comprising the steps of transfecting a lentivirus carrying a P4HA gene and a P4HB gene and a lentivirus carrying a COL3A1 gene into a host cell to construct the recombinant cell line;

[0010] The nucleotide sequence of the COL3A1 gene is shown in SEQ ID NO.1;

[0011] The nucleotide sequence of the P4HA gene is shown in SEQ ID NO.2;

[0012] The nucleotide sequence of the P4HB gene is shown in SEQ ID NO.3;

[0013] The host cell is 293F cell.

[0014] Furthermore, the method for constructing the lentivirus carrying the P4HA gene and the P4HB gene comprises the following steps:

[0015] The P4HA gene and the P4HB gene are constructed into a lentiviral overexpression vector to obtain a recombinant expression vector, and the recombinant expression vector is packaged with a virus to obtain the lentivirus carrying the P4HA gene and the P4HB gene.

[0016] Furthermore, the lentiviral overexpression vector is a pLVX-IRES-Puro vector.

[0017] Furthermore, the method for constructing the lentivirus carrying the COL3A1 gene comprises the following steps:

[0018] Constructing the COL3A1 gene into a lentiviral overexpression vector to obtain a recombinant expression vector, and packaging the recombinant expression vector to obtain the lentivirus carrying the COL3A1 gene; and / or

[0019] The recombinant expression vector is a pLVX-IRES-Hyg vector.

[0020] The present invention also provides a recombinant engineered cell constructed according to the above construction method and used for expressing triple helical structure type III recombinant human collagen.

[0021] The present invention also provides an application of the above-mentioned recombinant engineered cells in the preparation of triple helical structure type III recombinant human collagen.

[0022] The present invention also provides a method for preparing type III recombinant human collagen expressed by 293F cells, comprising the steps of obtaining the type III recombinant human collagen expressed by 293F cells by fermentation and purification using the above-mentioned recombinant engineering cells.

[0023] The present invention also provides a triple helical structure type III recombinant human collagen prepared according to the above preparation method.

[0024] The present invention also provides the use of the triple helical structure type III recombinant human collagen in the preparation of functional biomaterials, cosmetics or medical devices.

[0025] The present invention discloses the following technical effects:

[0026] The present invention constructs a cell line expressing type III recombinant human collagen by genetic engineering technology, and can realize the preparation of recombinant human collagen with a triple helix structure, self-assembly in vitro, and the same amino acid sequence as natural human collagen. The recombinant human collagen can be widely used in the fields of cosmetics, medical cosmetology, medical devices, biomedicine, etc., and has important market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 This is the WB test result of 293F-P4H cell line;

[0029] Figure 2 This is the SDS-PAGE detection result of 293F-P4H-COL3A1 cell line;

[0030] Figure 3 This is the SDS-PAGE test result of collagen sponge;

[0031] Figure 4 This is the circular dichroism detection result diagram. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1

[0038] 1. Sequence synthesis

[0039] 1.1 Codon optimization and gene synthesis of human COL3A1

[0040] The coding gene COL3A1 of human type III collagen α1 chain (α1(III) chain) was obtained by codon optimization, and its nucleotide sequence is shown in SEQ ID NO. 1. The α1(III) chain does not contain signal peptide, N-terminal propeptide and other parts that will be processed and shed in the α1(III) precursor protein.

[0041] SEQ ID NO.1:

[0042]

[0043] 1.2 Human P4HA codon optimization and gene synthesis

[0044] The coding gene P4HA of human proline hydroxylase was obtained by codon optimization, and its nucleotide sequence is shown in SEQ ID NO.2.

[0045] SEQ ID NO.2:

[0046]

[0047] 1.3 Human P4HB codon optimization and gene synthesis

[0048] The coding gene P4HB of human proline hydroxylase was obtained by codon optimization, and its nucleotide sequence is shown in SEQ ID NO.3.

[0049] SEQ ID NO.3:

[0050]

[0051] 2. Vector Construction

[0052] The synthesized COL3A1 gene (SEQ ID NO. 1) was constructed into the pLVX-IRES-Puro vector to obtain a recombinant overexpression vector (named COL3A1 in pLVX-IRES-Puro), which was then packaged into a lentivirus (named COL3A1-pLVX-IRES-Puro).

[0053] The company constructed the synthetic P4HA gene (SEQ ID NO.2) and P4HB gene (SEQ ID NO.3) into the pLVX-IRES-Hyg vector to obtain a recombinant overexpression vector (named P4HA-P4HB in pLVX-IRES-Hyg), and packaged it into a lentivirus (named P4HA-P4HB-pLVX-IRES-Hyg).

[0054] 3. Construction of prolyl-4-hydroxylase (P4H) cell line

[0055] 0.5 mL of lentivirus (P4HA-P4HB-pLVX-IRES-Hyg) was added to 2 mL of 293F cells. After 24 h, 500 μg / mL of Hyg was added to select the cells. When the cell viability was greater than 90%, samples were taken for WB detection. The protein expression results are shown in Figure 1 , and obtained the 293F-P4H cell line.

[0056] 4. Construction of collagen gene COL3A1 cell line

[0057] 0.5 mL of lentivirus (COL3A1-pLVX-IRES-Puro) was added to 2 mL of 293F-P4H cells. After 24 hours, 5 μg / mL of Puro was added to screen the cells to obtain the cell line 293F-P4H-COL3A1 expressing triple helical recombinant collagen. When the cell viability was greater than 90%, samples were taken for SDS-PAGE detection. The results are shown in Figure 2 .

[0058] The method for screening cells is as follows:

[0059] The cells were cultured to the logarithmic growth phase, counted by trypan blue staining, and diluted to about 250 cells / mL. 4 μL of cell suspension was aspirated and added to the middle of the wells of a flat-bottomed 96-well plate. Two columns of wells were added first, and the cells were observed under a microscope to see if they were monoclonal cells and marked. If the proportion of monoclonal cells in the two columns of wells was greater than 30%, the remaining wells were added at this concentration; if the wells were mostly polyclonal, the cells were diluted further according to the polyclonal situation in the wells, and two more columns of wells were added to observe the proportion of monoclonal cells until more than 30% of the wells were monoclonal; if no cells were seen in most wells, the cells were considered to be over-diluted, and the cells were diluted again from the previous concentration until more than 30% of the wells were monoclonal, and monoclonal cells were selected to obtain high-yield monoclonal cell lines.

[0060] 5. Cultivation and Purification

[0061] Take a monoclonal cell line in good condition for fermentation culture, and the inoculation density is 0.8×10 6 cells / mL, after 7 days of continuous culture, the cell viability dropped to 80%, and the fermentation broth was collected. Centrifugation was performed at 8000rpm and 4℃ for 10min, and the supernatant was collected as the triple helical recombinant collagen solution.

[0062] The triple-helical recombinant collagen solution was purified using Q Sepharose Fast Flow ion exchange chromatography. The loading buffer was 0.02 mol / L Tris-HCl buffer solution (pH 9.0±0.1). The loading volume was calculated based on the dynamic loading capacity of the Q Sepharose Fast Flow medium. After loading, the sample was washed with the loading buffer solution until the absorbance returned to the baseline. The eluted triple-helical recombinant collagen was collected and dialyzed into ddH2O, and lyophilized to obtain a collagen sponge. The protein purity was detected by SDS-PAGE, and the results are shown in Figure 3 The results showed that the protein purity was high.

[0063] 6. Circular dichroism detection

[0064] The collagen sponge was prepared into a 1 mg / mL aqueous solution, which was the recombinant collagen solution. Before the test, the recombinant collagen solution was balanced at 4°C for at least 24 hours. The wavelength scanning range was 190-260nm, with a step interval of 0.5nm and an average time of 5s. Each sample was scanned 3 times. The results are shown in Figure 4 The test results showed that the recombinant collagen had a triple helix structure.

[0065] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing a recombinant engineered cell for expressing triple helical type III recombinant human collagen, characterized in that: The method comprises the steps of transfecting a lentivirus carrying the P4HA gene and the P4HB gene and a lentivirus carrying the COL3A1 gene into a host cell to construct the recombinant cell line; The nucleotide sequence of the COL3A1 gene is shown in SEQ ID NO.1; The nucleotide sequence of the P4HA gene is shown in SEQ ID NO.2; The nucleotide sequence of the P4HB gene is shown in SEQ ID NO.3; The host cell is 293F cell.

2. The construction method according to claim 1, characterized in that: The method for constructing the lentivirus carrying the P4HA gene and the P4HB gene comprises the following steps: The P4HA gene and the P4HB gene are constructed into a lentiviral overexpression vector to obtain a recombinant expression vector, and the recombinant expression vector is packaged with a virus to obtain the lentivirus carrying the P4HA gene and the P4HB gene.

3. The construction method according to claim 2, characterized in that: The lentiviral overexpression vector is a pLVX-IRES-Puro vector.

4. The construction method according to claim 1, characterized in that: The method for constructing a lentivirus carrying the COL3A1 gene comprises the following steps: Constructing the COL3A1 gene into a lentiviral overexpression vector to obtain a recombinant expression vector, and packaging the recombinant expression vector to obtain the lentivirus carrying the COL3A1 gene; and / or The recombinant expression vector is a pLVX-IRES-Hyg vector.

5. A recombinant engineered cell constructed according to the construction method according to any one of claims 1 to 4 for expressing triple-helical type III recombinant human collagen.

6. Use of the recombinant engineered cell as claimed in claim 5 in the preparation of triple helical type III recombinant human collagen.

7. A method for preparing 293F cells expressing type III recombinant human collagen, characterized in that: The method comprises the steps of obtaining the 293F cells expressing type III recombinant human collagen by fermentation and purification using the recombinant engineered cells described in claim 5.

8. A recombinant human collagen with triple helical structure type III prepared according to the preparation method of claim 7.

9. Use of the triple helical type III recombinant human collagen as claimed in claim 8 in the preparation of functional biomaterials, cosmetics or medical devices.

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