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Nucleotide sequence of human inhibin A and recombinant expression method for human inhibin A

A nucleotide sequence and inhibin technology, applied in recombinant DNA technology, nucleic acid vectors, chemical instruments and methods, etc., can solve the problems of poor antibody specificity, gap between prokaryotic and eukaryotic expression systems, low activity of INHA protein, etc., to achieve Good stability and expression-promoting effect

Inactive Publication Date: 2019-12-10
普健生物(武汉)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although it can be used for polyclonal or monoclonal antibody preparation, the specificity of the prepared antibody is poor
Although prokaryotic expression systems are also available in the market to express in the periplasmic space, such as patent CN201480056265.1 (fusion construct of FAB-effector part of antiserum albumin and its preparation method), after all, prokaryotic and eukaryotic expression systems The gap is significant, and the activity of the expressed INHA protein is still very low

Method used

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  • Nucleotide sequence of human inhibin A and recombinant expression method for human inhibin A
  • Nucleotide sequence of human inhibin A and recombinant expression method for human inhibin A
  • Nucleotide sequence of human inhibin A and recombinant expression method for human inhibin A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Cloning and sequence analysis of human inhibin A

[0043] After analyzing and comparing the human inhibin A molecular sequence in Genbank, a large number of sequences in the nucleotide sequence of human inhibin A, such as signal peptide sequence, leader peptide sequence, enzyme cleavage site, G+C content and purification tag, etc. Information for sequence optimization design. The nucleotide sequence of the finally obtained human inhibin A is shown in Figure SEQ ID No.1, wherein the nucleotide sequence of the signal peptide nucleotide sequence is shown in SEQ ID No.2; the leader peptide nucleotide sequence The nucleotide sequence of the tag is shown in SEQ ID No.3; the nucleotide sequence of the purification tag is shown in SEQ ID No.4. The nucleotide sequence of the enzyme cutting site is NheI (the nucleotide sequence is gctagc) and XbaI (the nucleotide sequence is tctaga);

Embodiment 2

[0044] Embodiment 2: Construction of recombinant expression vector

[0045] This embodiment utilizes the nucleotide sequence of human inhibin A of embodiment 1 to construct a recombinant expression vector, the specific method is:

[0046] S1, designing primers for the nucleotide sequence of human inhibin A, specifically:

[0047] Upstream primer: 5'-ggagacccaagctggctagcgccgccaccatggtgctgcac-3';

[0048] Downstream primer: 5'-agcgggtttaaacgggccctctagattagtgatgatgatgatggtggctc-3';

[0049] S2. Establish a 10 μL PCR amplification system: take 5 μL of 2×PrimerSTARMAX, 0.4 μL of upstream primers, 0.4 μL of downstream primers, and 0.5 μL of templates, and add to 10 μL with double distilled water; the reaction conditions are 95°C pre-denaturation for 10 seconds , annealing at 55°C for 5s, extension at 72°C for 10s, 30 cycles, and then holding at 16°C; the amplified product was detected by 1% agarose gel electrophoresis, and the target fragment was recovered;

[0050] At this time,...

Embodiment 3

[0061] Embodiment 3: the preparation method of human inhibin A

[0062] Transfect the recombinant expression vector prepared in Example 2 into HEK293 cells, culture with serum-free medium, collect the medium by centrifugation, then filter it with a suction filter, place it on ice to prepare the target protein, or place it at -20°C Save it for future use.

[0063] 1 day before transfection, cells were passed to 0.5 × 10 6 / ml, the specific method is:

[0064] P1. One day before transfection, take the cells and filter them with a cell mesh, count them, and make the number of cells reach 0.5×10 6 / ml; Take 30ml and transfer to a new 125ml shaker flask;

[0065] P2. On the day of transfection, prepare two 2ml centrifuge tubes, add 0.8ml OPTI-MEM medium respectively; add 30μg recombinant expression vector to one tube, add 60μg PEI to the other tube, and gently invert the centrifuge tube several times to mix evenly;

[0066] P3. Add the diluted PEI to the diluted recombinant exp...

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Abstract

The invention discloses a nucleotide sequence of a recombinant-expression human inhibin A, and application of the nucleotide sequence. The application of the nucleotide sequence comprises the human inhibin A which is expressed by the nucleotide sequence, a preparation method for the recombinant-expression carrier of the human inhibin A, and a preparation method for the human inhibin A. The above method adopts artificial whole genes to synthesize the nucleotide sequence of the human inhibin A, a signal peptide nucleotide sequence, a leading peptide nucleotide sequence, a restriction enzyme cutting site, a G+C content, a purification tag and the like on the sequence are optimally designed to enable the nucleotide sequence to be more suitable for mammal cell expression; an INHA (inhibin A) gene sequence is synthesized by whole genes, and therefore, stability is good; a Kozak sequence is designed, expression can be accelerated, and the yield of the inhibin A is improved; and a His tag is designed so as to be favorable for purifying the expressed inhibin A.

Description

technical field [0001] The invention belongs to the technical field of human inhibin expression, in particular to a nucleotide sequence of human inhibin A and a method for recombinantly expressing human inhibin A. Background technique [0002] Inhibin is a glycoprotein hormone of the gonads and is a member of the transforming growth factors. Inhibin is derived from a macromolecular polypeptide of female ovarian granulosa cells and male testicular strut cells, which can strongly inhibit the secretion of follicle-stimulating hormone (FSH), but only slightly inhibit the secretion of LH. Inhibin is secreted by ovarian granulosa cells, and the complete inhibin molecule has a molecular weight of 32,000. It consists of two different subunits (a subunit and b subunit) connected by disulfide bonds. The a subunit can also be found in follicular fluid and serum. In addition, the free a subunit and b subunit have no biological activity. Inhibin can feedback inhibit the release of fo...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N15/85C07K14/575C07K14/47
CPCC07K14/4703C07K14/575C07K2319/02C07K2319/21C07K2319/50C12N15/85C12N2800/107
Inventor 李沛代腾飞秦伏波鲁亮万定一张永霞
Owner 普健生物(武汉)科技有限公司
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