CN103173474A - Gene sequence for expressing soluble recombinant human hyaluronidase PH20 in CHO (Chinese hamster ovary) cell - Google Patents

Abstract

The invention discloses a gene sequence for expressing soluble recombinant human hyaluronidase PH20 by a CHO (Chinese hamster ovary) cell. The gene sequence has a nucleotide sequence shown in SEQ ID NO.2; after a kozak consensus sequence is added to 5' terminal of the optimized gene sequence, the optimized gene sequence is connected to pOptiVEC-TOPO carrier, and stably transferred to the Chinese hamster ovary cell improved by genetic engineering, so as to achieve high expression of the soluble recombinant human hyaluronidase PH20 in a mammalian cell.

Description

A kind of gene order for expressing cho cell soluble recombined human Unidasa PH20

Technical field

The invention belongs to biological technical field, be specifically related to a kind of gene order and the engineering cell strain that contains this gene for Chinese hamster ovary cell (Chinese hamster ovary celI) recombinant expression of soluble Unidasa.

Background technology

Unidasa is the hyaluronic enzyme family of degraded, and hyaluronic acid is the essential component of extracellular matrix and the main component of a matter barrier.Unidasa can make hyaluronic acid produce degraded effect, thereby reduces hyaluronic activity and viscosity, improves liquid infiltration ability in tissue.

The hyaluronic acid enzyme family comprises the bacterium Unidasa, from the Unidasa of parasite and crustacean with from mammiferous Unidasa.And 6 hyaluronic acid-like enzyme genes are arranged in people's genome: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and PH20/SPAM1.Wherein, HYAL3, HYAL4 present not to be had or the activity of degraded hyaluronic acid seldom, and HYAL1, HYAL2 are the acid activity Unidasas, the general catalytic activity that lacks under condition of neutral pH, PH20 is neutral organized enzyme, has catalytic activity in the pH scope of the blood of human body or tissue juice.

Unidasa is mainly used to improve the infiltration of other injectable drugs and increases the tissue permeability of other drug and promote diffusion or disperse.Modal application is the rapid osmotic (as dentistry) of ophthalmologic operation and local anesthetic, uses in the time of can't finding blood vessel in the child vein drop.Also can be used for treating tumour, coordinate subcutaneous injection to replace intravenous drip with the antibody of costliness, keep the purposes such as unobstructed of Regular Insulin and other pump administration.The Unidasa immunogenicity of extracting in animal tissues is stronger owing to deriving from, and is unsuitable for applying to the human clinical, and utilizing the genetically engineered recombinant technology to express, produce people's Unidasa has become inevitable trend.Because the C of people's total length PH20 aminoacid sequence end contains glycosyl-phosphatidyl inositol (GPI) decorating site, affected its solubility secretion outside host cell, therefore will remove encoding sequence corresponding to PH20 precursor protein (precursor) aminoacid sequence C end when building the strain of solubility PH20 secreting, expressing, and stay encoding sequence corresponding to 1-482 amino acids in the PH20 precursor protein.The albumen of expressing is called soluble recombined human PH20 albumen (soluble rHuPH20).As the Hylenex Unidasa of drugs approved by FDA listing, be namely soluble rHuPH20.

Chinese hamster ovary cell (Chinese hamster ovary cell, CHO) is a kind of fibroblast that derives from Chinese hamster ovary, is mammalian expression system host cell commonly used.It has lot of advantages: post transcriptional modificaiton function accurately, the glycosylation pharmaceutical protein of expressing aspect molecular structure, physicochemical property and biological function near the native protein molecule, the expression product cell exocrine, be convenient to separation and purification, and because it belongs to inoblast, seldom secrete intrinsic protein, utilize the separation and purification of foreign protein.

We find under study for action, and the level that the engineering cell strain that builds with the genetically engineered Chinese hamster ovary celI strain (being specially the DG44 cell strain) of carrier transfection that contains solubility rHuPH20 natural gene sequence is expressed PH20 is lower, causes production cost high.We can improve the expression level of target protein in the Chinese hamster ovary celI strain greatly at the gene order after to native sequences optimization.

Summary of the invention

The object of the invention is to: the low defective of expressing of natural gene for coding solubility rHuPH20, by the optimized gene sequence, realize the high expression level of solubility rHuPH20 in the CHO engineering cell strain.

The technical solution used in the present invention is:

The gene order of the people's Unidasa PH20 that optimizes, it has the nucleotide sequence shown in SEQ ID NO.2.

Add the kozak consensus sequence at above-mentioned people's Unidasa PH20 optimized gene sequence 5 ' end, obtain its derivative DNA sequence dna

A kind of cloning vector contains above-mentioned people's Unidasa PH20 optimized gene sequence or by its derived dna sequence.

A kind of expression vector contains above-mentioned people's Unidasa PH20 optimized gene sequence or by its derived dna sequence.

The initial vector of described expression vector is mammalian cell expression vector.

A kind of Chinese hamster ovary cell expression strain contains above-mentioned expression vector.

Described Chinese hamster ovary cell expression strain is the DHFR gene defection type.

Above-mentioned Chinese hamster ovary cell expression strain is in the application of producing on soluble recombined human Unidasa PH20.

The invention has the beneficial effects as follows: the gene order of the PH20 of the present invention after optimizing, be connected to the pOptiVEC-TOPO carrier and complete plasmid construction, after being stably transfected into genetically engineered Chinese hamster ovary cell, significantly improve soluble recombined human Unidasa PH20(soluble rHuPH20 with former sequence phase specific energy) expression level.

Description of drawings

Fig. 1 is the solubility rHuPH20 gene order optimized of the present invention and the aminoacid sequence comparative analysis of natural gene sequence encoding;

Fig. 2 is pOptiVEC-TOPO carrier structure schematic diagram.

Embodiment

The present invention is further illustrated below in conjunction with embodiment, but be not limited to this.

Due to the detection that will be referred to hyaluronidase activity in following operation and PH20 gene copy number quantitatively, so first introduce the operation of these two kinds of methods here.

One, the activity test method of Unidasa (nephelometry)

1, nephelometry principle: the hyaluronic acid of acidifying (HA) solution can form stable colloidal solution with serum, after hyaluronic acid decomposes, will become clarification with the mixed solution of serum.Because Unidasa has the effect that the catalysis hyaluronic acid decomposes, therefore within the specific limits, hyaluronidase activity and reacted turbidity are inversely proportional to.

2, material: horse serum and HA reference substance are all from SIGMA company.HA is configured to 0.5U/mL, 1U/mL, 2U/mL, 4U/mL, 6U/mL, 8U/mL, 10U/mL totally 7 activity unit's reference substances;

3, concrete testing process: HA solution adds in 96 orifice plates, and then every hole 20 μ L add 20 μ L reference substance or sample liquid, abundant mixing, and 37 ℃ of water-bath 30 min, after taking out, every hole adds 160 μ L horse serums, mixing, room temperature is placed 30 min, surveys A ₆₄₀With absorbancy ABe ordinate zou, tire (U) of reference substance solution is X-coordinate, drawing standard curve.The sample absorbancy that records is calculated activity according to typical curve.

Two, express the quantitative PCR detecting method of solubility rHuPH20 gene copy number in strain

1, design of primers and synthetic

Design respectively primers F 1/R1, F2/R2 according to people's Unidasa PH20 gene order natural and that optimize, according to mouse house-keeping gene β-actin gene order design primer β-actin F/R, see Table 1.

2, extract CHO DG44 genomic dna and recombinant plasmid

Extract each recombinant cell strain genome, CHO DG44 genomic dna and recombinant plasmid, with DNA quality and the concentration of nucleic acid-protein analyser Detection and Extraction.

3, standard substance build

Calculating contains 1,5,25,125,625 required expression vector quality of copy number of foreign gene, and CHO DG44 genome quality is got 10ng, and formula is as follows:

[the expression vector size (N bp) of copy number * contain PH2O gene]/[CHO DG44 group DNA single times body size (3 * 10 ⁹Bp)]=contain transgene expression vector quality/CHODG44 genome quality

The expression vector (representing different copy numbers) of different mass is mixed with CHO DG44 genomic dna (10 ng), build standard substance.

4, typical curve determines

Turn the PH20 gene fragment with PH2O primers F/R amplification, with the amount of β-actin F/R amplification house-keeping gene β-actin as internal reference mark genomic dna.The amplified production of PH20 F1/R1 primer is 445 bp; The amplified production of PH20 F2/R2 primer is 445 bp; The amplified production 200bp of β-actin F/ R primer.The PH20 gene fragment is detected the amplification Ct that primer amplification Ct (PH20) deducts corresponding β-actin gene (β-actin), the △ Ct that obtains, then the logarithmic value mapping of the copy number of sample is obtained the typical curve of absolute quantitation.

5, real-time quantitative PCR reaction system and condition

Reaction system is: template 2 μ L, upstream primer (10 pmolL ^-1) 0.8 μ L, downstream primer (10 pmolL ^-1) 0.8 μ L, SYBR Green Real-Time PCR Mix 10 μ L, distilled water 6.4 μ L.

Reaction conditions is: 95 ℃ of 10 min; 95 ℃ of 10 s, 60 ℃ of 20 s, 72 ℃ of 30 s, 32 circulations.Each sample arranges 3 Duplicate Samples, gets average.

6, recombinant cell strain solubility rHuPH20 gene copy number is identified

Extracting each recombinant cell strain genome, is 0.8% agarose gel electrophoresis and A260/A280 ultraviolet detection through volume fraction, shows that genome pollutes without protein and RNA.Get respectively 10 ng and carry out real-time quantitative PCR, amplification PH20 gene and β-actin gene, and 3 independent repeated experiments draw each recombinant cell strain about the △ Ct value of PH20 gene.Copy number calculates according to formula corresponding to typical curve.

Embodiment

One, the optimization of the gene order of soluble human hyaluronidase enzyme PH20

On the basis of analyst's Unidasa PH20 natural gene sequence (nucleotide sequence is as shown in SEQ ID NO.1), eliminate rare codon, remove unstable motif, removal RNA loop-stem structure and utilize the best codon of Chinese hamster ovary cell to redesign.Gene order after optimization is as shown in SEQ ID NO.2.Gene order after optimizing and native sequences are carried out the nblast homology compare, do not draw the result that both have homology; And carry out homology relatively with tblastx, show the aminoacid sequence 100% homology (see figure 1) that both encode.

For improving the expression efficiency of this gene order in eucaryon host, at gene order coded amino acid position 1(methionine(Met)) add Kozak consensus sequence GCCACC before corresponding codon.Gene order after optimization is synthetic by English Weihe River company of prompt base (Shanghai) trade Co., Ltd.

Two, comprise the screening of the height copy/overexpression cell line of optimized gene

1, the structure of carrier for expression of eukaryon (PH20-pOptiVEC – TOPO)

See Table 1 with PCR primers F 2 and R2() the synthetic PH20 optimized gene (containing the kozak sequence before the coding region) of amplification, the gene fragment of amplification is connected to carrier for expression of eukaryon pOptiVEC-TOPO(with the T-A cloning and sees Fig. 2) in, build and obtain recombinant expression vector PH20-pOptiVEC – TOPO.

2, recombinant expression vector transfection CHO-DG44 cell

PH20-pOptiVEC – TOPO recombinant vectors electroporation transfection CHO-DG44 cell.Wherein, CHO-DG44 cell used is the DHFR gene defection type, does not contain dihydrofolate reductase gene, can not nucleic acid, must grow in the substratum that contains xanthoglobulin (H) and thymidine (T).

Screening positive cell principle is: when the goal gene of transfection was connected with the dhfr gene, positive cell had also just obtained the dhfr gene.Can grow in the substratum that lacks HT.

Transfection process is as follows:

With CD OptiCHO culture medium culturing amplification CHO DG44 cell, carry out cell counting before transfection, guarantee that 95% is viable cell.

Centrifugal collection 6 * 10 ⁷Cell precipitation, and at 2 * transfection damping fluid (40mM HEPES, pH7.0,274mM NaCl, 10Mm KCl, the 1.4mM Na of 0.7mL ₂HPO ₄, 12mM dextran) in resuspended one-tenth 2 * 10 ⁷The density of cell.Add 90 μ L(250 ug thereafter) use ClaIThe recombinant plasmid of restriction endonuclease linear process, and cell/plasmid solution is transferred in the electroporation cuvette in room temperature.The mixture of cell/plasmid carries out electroporation under the condition of the electrical condenser of 875V/cm and 960uF energising processes.

3, the cell of screening stable transfection

In order to obtain the cell strain of high expression level Unidasa PH20, be by the cell of a collection of stable transfection success of screening, namely the pOptiVEC – TOPO Vector with goal gene has successfully inserted cell chromosome.Process is as follows:

After electroporation, cell is shifted out from cuvette and transfer in the CD OptiCHO substratum of 5mL, and make them in 6 porocyte culture plates, in 5% CO ₂In 37 ℃ in the incubator of humidification without the growth of selecting 2 days.Get supernatant after 2 days and detect Unidasa content with nephelometry.Collecting cell counts and uses the CD OptiCHO substratum of xanthoglobulin (H), thymidine defective (T) to be diluted to 2 * 10 ⁴Individual viable cell/mL.Cell suspension is transferred to 96 porocyte culture plates by the 0.1mL/ hole, adds H, T defective substratum to 0.2mL.Then at 5%CO ₂, cultivated 3-4 days in the incubator of 37 ℃.Collect every hole supernatant and detect Unidasa content with nephelometry.Select 5 the highest clones of expression amount, increase with the substratum of H, T defective, be settled to 5 * 10 with CD OptiCHO substratum after collecting cell ⁵Individual viable cell/mL, and frozen processing.

4, with Rheumatrex (MTX) amplification, the high copy/high expression level monoclonal cell of screening

(1) MTX carries out gene amplification

The operation of carrying out gene amplification with MTX is as follows:

Carries out amplification cultivation with CD OptiCHO substratum after freeze-stored cell recovery, centrifugally remove old substratum, with 3 * 10 ⁵/ mL cell density is inoculated in the CD OptiCHO substratum that 100-300mL contains 50nM MTX.Enchylema is transferred in the culturing bottle of 0.5-1.0 L, in 5%CO ₂, culturing cell in the incubator environment of 37 ℃.Every 3-4 days passage cell is in fresh substratum, and inoculum density is 2 * 10 ⁵-3 * 10 ⁵Cell/mL.When the active cells number begins to increase, the beginning passage cell, inoculum density is 2 * 10 ⁵-3 * 10 ⁵Cell/mL.When viable count greater than 90% the time, freeze-stored cell.

(2) limiting dilution assay screening cell clone

In limiting dilution assay, cell substratum used is for cloning substratum fully, from Invitrogen company.100mL clones fully that in substratum, composition is: 86mL CD FortiCHO; The freshly prepared 200mM L-glutamine of 3mL; 10mL conditioned medium; 1mL 100 * HT mother liquor.

The limiting dilution assay operation steps is as follows:

1. prepare the centrifuge tube of several 50mL, the accurate calculation cell density, the cell of getting the 5-10mL stable transfection is added to centrifuge tube 1, then doubling dilution to 1000 cell/mL.

2. substratum is cloned in preheating fully, and absorption 200 μ L cell suspensions from dilution tube (1000 cells/mL) to cloning in substratum fully.Reverse 5-6 time back and forth, transfer to sterile chamber.The cell that is divided into 200 μ L dilutions is in 96 orifice plates in culture plate.Under 37 ℃ of conditions, cultivated 96 orifice plate 10 days.Clone with microscope observing cell after 10 days.Nephelometry detects the cell expressing situation, and the expression amount and the copy number results that detect see Table 2, filter out 5 high-expression clone cell strains.As known from Table 2, under the condition of similar copy number, the expression amount that the PH20 gene pairs of optimizing in the present invention is answered will be significantly higher than the expression amount that natural PH20 gene pairs is answered.

Annotate: ¹Compare expression amount difference very remarkable (p＜0.01) for organizing with natural PH20

Only for introducing preferred case of the present invention, to those skilled in the art, any apparent changes and improvements of carrying out in the scope that does not deviate from spirit of the present invention all should be regarded as a part of the present invention to above embodiment.

＜110〉Community in Baiyunshan, Guangzhou is visitd enlightening biological medicine company limited

＜120〉a kind of gene order for expressing cho cell soluble recombined human Unidasa PH20

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<170> PatentIn version 3.5

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Claims (8)

1. the gene order of the people's Unidasa PH20 that optimizes, it has the nucleotide sequence shown in SEQ ID NO.2.

2. by the derivative DNA sequence dna of the optimized gene sequence of claim 1, it is characterized in that, add the kozak consensus sequence at optimized gene sequence 5 ' end.

3. cloning vector, it contains gene order claimed in claim 1 or DNA sequence dna claimed in claim 2.

4. expression vector, it contains gene order claimed in claim 1 or DNA sequence dna claimed in claim 2.

5. expression vector according to claim 4, is characterized in that, initial vector is mammalian cell expression vector.

6. Chinese hamster ovary cell expression strain, it contains the described expression vector of claim 4 or 5.

7. Chinese hamster ovary cell expression strain claimed in claim 6 is the DHFR gene defection type.

8. Chinese hamster ovary cell expression strain claimed in claim 6 is in the application of producing on soluble recombined human Unidasa PH20.

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