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Recombinant humanized III-type collagen and prokaryotic expression method thereof

A collagen and expression carrier technology, applied in the field of recombinant human type III collagen and its prokaryotic expression, can solve the problems of not being able to copy human collagen well, achieve good biological activity and promote cell migration

Active Publication Date: 2020-05-01
河北纳科生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above has the following defects: the above-mentioned patent selects a very short fragment as a repeating unit, spliced ​​together to express a long fragment of recombinant human collagen, which cannot replicate the sequence of human collagen well

Method used

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  • Recombinant humanized III-type collagen and prokaryotic expression method thereof
  • Recombinant humanized III-type collagen and prokaryotic expression method thereof
  • Recombinant humanized III-type collagen and prokaryotic expression method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 gene design and synthesis

[0031] (1) Gene design: According to the amino acid sequence of human type III collagen (UniProtKB / Swiss-Prot:P02461.4), SEQ ID NO: 1:

[0032] MYDSYDVKSGVAVGGLAGYPGPPGPPPGPAGPPGPPGPPGTSGHPGSPGSPGYQGPPGEPGQAGPSGPPGPPGAIGPSGPAGKDGESPGGRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGVGAAAIAGIGGEKAGGFAPYYHHHHHH

[0033] Use the online design tool Jcat (http: / / www.jcat.de / ) to reverse design the gene sequence, aiming at the preferred codons required for expression in the host Escherichia coli, and remove the NdeI and XhoI restriction sites during the design process, which is beneficial to the later stage Gene manipulation, the optimized gene sequence is shown as SEQ ID NO: 2, compared with the...

Embodiment 2

[0038] Example 2 Construction of recombinant expression vector pET30-1230

[0039] Use NdeI and XhoI double enzymes to digest the gene SEQ ID NO:2 fragment obtained in Example 1. The enzyme digestion system is as follows: 50 μL of enzyme digestion system, 5 μL (2 μg) of SEQ ID NO:2 fragment, 1 μL each of NdeI and XhoI enzymes, buffer 5 μL solution, supplemented with 50 μL sterilized double distilled water, and incubated at 37°C for 4 hours. Use the same system to digest plasmid PET30a (+). The linear fragment was obtained by enzyme digestion, the target fragment was purified by DNA gel recovery kit, and the obtained fragment was ligated with T4 ligase. The ligation system was as follows: T4 DNA ligase 1 μL, 1230 digested fragment 3 μL, PET30a (+) 2 μL of digested fragments, 1 μL of ligation buffer, and 10 μL of sterilized double-distilled water. Incubate at 16°C for 4h. Transform the incubated product into host bacteria by heat shock method E. coli In DH5α, apply to LB cu...

Embodiment 3

[0040] Example 3 Construction of Engineering Bacteria

[0041] Pick a single colony of Escherichia coli BL21 (DE3) and inoculate it in an LB test tube, then shake overnight at 37°C; add 0.5mL overnight culture solution to a Erlenmeyer flask containing 50mL LB, and shake vigorously at 37°C for about 2 hours to grow the bacteria to In the early logarithmic period; under sterile conditions, transfer the bacteria to a 50mL polypropylene tube pre-cooled with ice, and place it on ice for 10 minutes; centrifuge at 4°C, 4000rpm, pour off the supernatant, and invert the tube to make the residual liquid flow out as much as possible; add 6mL 0.1mol / L CaCl pre-cooled with ice 2 Resuspend the pellet and place it on ice for 30min; centrifuge at 3000rpm at 4°C, pour off the supernatant, invert the tube to make the residual liquid flow out as much as possible; add 1.2mL of 0.1mol / L CaCl pre-cooled with ice 2 Resuspend the pellet (if you want to prepare competent cells for storage at -70°C, a...

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PUM

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Abstract

The invention relates to a recombinant human III-type collagen and a prokaryotic expression method thereof. The amino acid sequence of the humanized III-type collagen is shown as SEQ ID NO.1, and thenucleotide sequence of an encoding gene is shown as SEQ ID NO.2. The method comprises the following steps: carrying out codon optimization and design to obtain the gene sequences; inserting the gene sequences into a position between enzyme cutting sites NdeI and XhoI of an expression vector PET30a(+) to construct a recombinant expression vector pET30-1230; transforming Escherichia coli BL211(DE3)competent cells; selecting positive clones; and performing culture induction for efficient expression.

Description

technical field [0001] The invention belongs to the technical field of optimized coding genes, and in particular relates to a recombinant human type III collagen and a prokaryotic expression method thereof. Background technique [0002] Collagen is a kind of glycoprotein, which can be divided into more than 28 types of collagen according to different tissue parts, physiological functions, and molecular structures. Type I, type II, and type III collagen account for the largest proportion and the most thorough research. Collagen in human skin is type I collagen and type III collagen, type I collagen mainly exists in adult skin, tendon, bone tissue, type III collagen mainly exists in infant skin or vascular intima, intestinal tract, type I collagen and type III Collagen is closely related to the skin damage repair process and repair quality. Among them, type III collagen is a triple helix formed by three peptide chains twisted to the right, which has excellent biological activ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/12C12N15/70C12N15/66C12N1/21C12R1/19
CPCC07K14/78C12N15/66C12N15/70
Inventor 徐兰举齐磊刘鑫申翠美杜亚东
Owner 河北纳科生物科技有限公司
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