CN104611340A - Recombinant human CC16 gene, construction of eukaryotic expression vector of CC16 gene, and purification of recombinant protein - Google Patents

Abstract

The invention provides a synthesized recombinant human CC16 gene shown as SEQ ID NO.1, construction of a eukaryotic expression recombinant plasmid containing the CC16 gene, and production of purified recombinant human CC16 protein after cell transfection. The obtained recombinant human CC16 protein can inhibit PLA2 activity, so that a condition is created by using the recombinant protein to realize intervention therapy on inflammatory lung diseases.

Description

The structure of recombinant human CC16 gene, its carrier for expression of eukaryon and the purifying of recombinant protein

Technical field

The invention belongs to gene engineering technology field, relate to a kind of people CC16 gene utilizing DNA recombinant technology to synthesize, and the separation and purification after the structure of Eukaryotic expression recombinant plasmid and expression of recombinant proteins.

Background technology

Clara cell 10kDa protein matter is primarily of the fibre-less columnar epithelial cell on bronchiole, bronchioli terminales mucous membrane---and Clara emiocytosis produces, because its molecular weight is about 15.8kDa, therefore be called Clara cell 10kDa protein, be also called Clara cell secretory protein (Clara cell secretory protein, CCSP).CC16 is tissue specific protein, mainly expresses in lung tissue, accounts for 2% ~ 3% of soluble proteins in the bronchoalveolar lavage fluid (BALF) of people.

Research shows, the generation of many chronic pulmonary inflammatory diseases all reduces relevant with lung Clara Leukopenia and CC16 content, as chronic obstructive pulmonary disease (COPD), asthma etc.COPD is that its pathogenesis is not yet completely clear with the flow limitation of the Progressive symmetric erythrokeratodermia development chronic respiratory tract disease that is feature, and inflammatory reaction caused by smoking and bacteriological infection is the Major Risk Factors of COPD morbidity.In smoker's bronchiolar epithelium, Clara cell quantity obviously reduces, and in bronchoalveolar lavage fluid and serum, CC16 content also obviously reduces.Pilette C etc. study discovery, compare, Clara Leukopenia in COPD patient small airway with Healthy People, and cause CC16 to secrete and reduce, in BALF, CC16 content reduces.In rat COPD model prepared by smoke from cigarette, also been observed identical result, when finding that airway inflammation occurs, Clara cell and CC16 obviously reduce, and CC16 minimizing can cause air flue anti-inflammatory power to decline, and causes inflammation to worsen further.And after giving Antioxidant N-acetyl-cysteine, suppress Airway inflammatory response by the synthesis of the recovery and CC16 that promote Clara cell and secretion, the inflammation of COPD can be alleviated.

As endogenic anti-inflammatory substance, CC16 has very important immunomodulatory and anti-inflammatory action in lung, and one of its anti-inflammatory mechanisms suppresses phosphide enzyme A ₂(PLA ₂) activity.PLA ₂be the active enzyme protein being present in nearly all karyocyte, be divided into the PLA of the secretor type relying on calcium ion ₂(sPLA ₂) and endochylema type PLA ₂(cPLA ₂) and non-calcium dependent form PLA ₂(iPLA ₂).PLA ₂being the hydrolase of specific glyceryl phosphatide Sn-2 position fat key, is signaling molecule important in cell.PLA ₂being the rate-limiting enzyme producing lipid medium and platelet activation factor (PAF), is also the activation object of inflammatory cytokine.Intracellular toxin can increase PLA ₂activity, and then aggravates inflammation reaction.Although as the CC16 of the endogenous anti-inflammatory factor, the concrete molecular mechanism of its anti-inflammatory still imperfectly understands, and in vitro study confirms, CC16 is by suppressing PLA ₂activity, restriction arachidonic acid metabolism and take leukotriene as the cascade reaction of mediated factor, thus suppress the synthesis of prostaglandin(PG) and interleukin.

Therefore, supplement CC16 to make up the anti-inflammatory factors lacked in chronic inflammation disease process, intervention effect can be played to the generation development of disease, just highly pay close attention to.And adopt the eukaryon expression plasmid of gene overexpression technique construction CC16, making it to express Clara cell 10kDa protein in pulmonary epithelial cells, supplement CC16 not enough in lysis, is effective trial of the pulmonary inflammation diseases such as treatment COPD.

Summary of the invention

The object of this invention is to provide a kind of recombinant human CC16 gene, and build its Eukaryotic expression recombinant plasmid, Restruction people Clara cell 10kDa protein matter after transfectional cell purifying, thus create conditions for utilizing this recombinant protein confrontation inflammatory lung disease to carry out Clinical intervention.

The present invention is according to the cDNA complete genome sequence of people's Clara cell 10kDa protein matter, and design and synthesis recombinant human CC16 gene, its nucleotide sequence, as shown in SEQ ID NO.1, adds Kazak sequence and 3 × Flag label respectively at the two ends of gene.

Invention also provides a kind of Eukaryotic expression recombinant plasmid containing above-mentioned recombinant human CC16 gene, employ carrier for expression of eukaryon pMSVpuro, its with xhoi and ecoRi restriction enzyme site, will be through xhoi and ecoRthe recombinant human CC16 gene of I double digestion is connected with described carrier for expression of eukaryon pMSVpuro, forms Eukaryotic expression recombinant plasmid of the present invention, has the nucleotide sequence shown in SEQ ID NO.2.

Present invention also offers a kind of host cell comprising described Eukaryotic expression recombinant plasmid, described Eukaryotic expression recombinant plasmid is proceeded in HEK 293T cell, construct the eukaryotic cell expression system containing described Eukaryotic expression recombinant plasmid.

Present invention also offers the method for producing described recombinant human Clara cell 10kDa protein in HEK 293T cell expression system, comprise the following steps:

1) to verify the nucleotide sequence of answering according to people's Clara cell 10kDa protein, synthesizing recombined human CC16 gene;

2) recombinant human CC16 gene is connected in carrier for expression of eukaryon pMSVpuro, builds the Eukaryotic expression recombinant plasmid pMSCVpuro-hCC16 containing described recombinant human CC16 gene;

3) with described Eukaryotic expression recombinant plasmid pMSCVpuro-hCC16 transfection HEK 293T cell, the HEK 293T cell expression system containing recombinant human CC16 gene is built;

4) in described HEK 293T cell expression system, culture expression recombinant human Clara cell 10kDa protein under serum free medium system;

5) also recombinant human Clara cell 10kDa protein described in purifying is separated.

Wherein, described step 2) in carrier for expression of eukaryon pMSCVpuro warp xhoi and ecoRconnect with the same recombinant human CC16 gene through double digestion after I double digestion.

In the present invention, described HEK 293T cell is containing 10% new-born calf serum, 100u/ml penicillin, in the DMEM high glucose medium of 100ug/ml Streptomycin sulphate, and 37 DEG C and 5%CO ₂grow under condition, within 3 ~ 4 days, change liquid once.

Further, in the method for described Restruction people Clara cell 10kDa protein, described step 3) in, add in transfection the tetracycline that final concentration is 1.5 μ g/ml after 24 hours, cell cultures 48 hours, to filter out the cell clone of stably express.

Further, described step 5) separation and purifying specifically comprise the following steps:

Collect step 4) serum free medium supernatant;

Remaining cell PBS washs, and obtains the cell of stable transfection pMSCVpuro-hCC16, hatches 30min with protein lysate, collecting cell lysate;

By centrifugal to cell pyrolysis liquid and the serum-free medium collected, collect supernatant, add at 4 DEG C in advance with the ANTI-FLAG M that TBS balances ₂in affinity gel, make albumen and gel overnight incubation;

Wash the non-associated proteins on affinity gel off with TBS after, then with the target protein that elution affinity gel combines, add 0.5M Tris HCl in elutriant, pH7.4; 1.5M NaCl, uses the evaporating pipe of molecular weight cut-off 3kDa to concentrate target protein, obtains the recombinant human Clara cell 10kDa protein of purifying.

Wherein, described protein lysate contains 50mM Tris-HCl, pH7.4; 150mM NaCl; 1mM EDTA; 1%TRITON X-100; Described TBS contains 50mM Tris HCl, 150mM NaCl, pH7.4; Described elutriant is 0.1M glycine-HCl, pH3.5.

The present invention has synthesized modified recombinant human CC16 gene, 3 × Flag sequence label is added at its carboxyl terminal, and connect in carrier for expression of eukaryon pMSCVpuro, successfully construct Eukaryotic expression recombinant plasmid pMSCVpuro-hCC16, and make people CC16 gene successful expression in HEK 293T cell.

The present invention adopts the method for affinity purification, obtains the recombinant human Clara cell 10kDa protein that purity is greater than 99%, reaches industrial requirement.

The recombinant human Clara cell 10kDa protein that the present invention obtains can suppress PLA ₂activity, illustrate that the recombinant protein obtained has biologic activity, this is just the anti-inflammatory properties of research recombinant human Clara cell 10kDa protein, and the intervention effect particularly in pulmonary inflammation disease is laid a good foundation.

Accompanying drawing explanation

Fig. 1 is that the PCR of Eukaryotic expression recombinant plasmid pMSCVpuro-hCC16 of the present invention identifies agarose gel electrophoresis figure.

In figure, M:DNA molecule marker; 1 ~ 3:pMSCVpuro-hCC16; 4: negative control.

Fig. 2 is the agarose gel electrophoresis figure of Eukaryotic expression recombinant plasmid pMSCVpuro-hCC16 double digestion product of the present invention.

In figure, M:DNA molecule marker; 1 ~ 2:pMSCVpuro-hCC16 digestion products.

Fig. 3 is the Western blotting detection figure that recombinant human Clara cell 10kDa protein of the present invention is expressed in host cell.

In figure, 1:pMSCVpuro-hCC16 plasmid transfection albumen; 2:pMSCVpuro plasmid transfection albumen.

Fig. 4 is the SDS-PAGE detection figure of the recombinant human Clara cell 10kDa protein of purifying.

Fig. 5 is that recombinant human Clara cell 10kDa protein is to PLA ₂active inhibition.

Embodiment

The structure material used in the embodiment of the present invention 1 ~ 5 and the source of main agents.

Carrier for expression of eukaryon: pMSVpuro, purchased from American Clontech company.

HEK 293T cell, purchased from Beijing Union Medical College basis institute cell centre.

Escherichia coli Top10,2 × Taq PCR Master Mix, T ₄dNA ligase, purchased from Beijing Quanshijin Biotechnology Co., Ltd; DL 2000 DNA Marker, sepharose reclaim test kit, the little extraction reagent kit of plasmid, RIPA protein extract, BCA protein quantification test kit, purchased from the vast Tai Heng Bioisystech Co., Ltd in Beijing; The anti-human CC16 polyclonal antibody of rabbit, purchased from American Abcam company; The anti-Flag monoclonal antibody of people, the anti-β of people-actin monoclonal antibody, purchased from Hua Da gene company limited; Restriction enzyme ecoRi and xhoi, spin (Shanghai) bio tech ltd purchased from Japan; Albumen Marker, purchased from Shanghai Bo Gu Bioisystech Co., Ltd; Goat anti human (rabbit) IgG of horseradish enzyme labelling, purchased from mountain gold bridge company limited in Beijing; Entranster-D transfection reagent, ECL luminescent solution, purchased from Beijing English lattice benefactor department; M ₂flag-beads purchased from American sigma company.

Other reagent are domestic analytical pure.

Embodiment 1

Adopt the method based on PAS (PCR-based Accurate Snthesis); according to people's Clara cell 10kDa protein matter encoding gene (gene accession number: U01101.1); design recombinant human CC16 gene order; synthetic gene total length splicing primer; each design protection base at primer two ends; 5 ' end adds GCCACC (Kazak sequence), and 3 ' end adds 3 × Flag label.Gene order is synthesized by Nanjing bronze object Bioisystech Co., Ltd, and the hCC16 gene of synthesis has the nucleotide sequence shown in SEQ NO.1.

By pMSCVpuro carrier and hCC16 gene order restriction enzyme xhoi and ecoRi double digestion, agarose gel electrophoresis is separated enzyme and cuts after product.Digestion products reclaims after test kit reclaims through gel, enzyme is cut carrier and enzyme cut hCC16 with 1: 3 mixed in molar ratio, at T ₄under the effect of DNA ligase, 22 DEG C of reaction 1h.Then will connect product conversion competent cell Top10, the LB be spread evenly across containing penbritin (100 μ g/ml) is dull and stereotyped, 37 DEG C of incubated overnight.Picking mono-clonal bacterium colony, is inoculated in the LB liquid medium containing penbritin and increases in a large number.

Adopt respectively bacterium liquid PCR, xhoi and ecoRi double digestion and DNA sequence dna sequencing analysis carry out positive clone identification.PCR reaction system is: template 2 μ l, and upstream and downstream primer (10 μm of ol/L) each 1 μ l, 2 × Taq Master Mix 25 μ l, adds deionized water to 50 μ l.Reaction conditions: 94 DEG C 5 minutes; 94 DEG C 15 seconds, 56 DEG C 15 seconds, 72 DEG C 15 seconds, totally 30 circulations; 72 DEG C 3 minutes.Negative control group (replacing bacterium liquid with water in reaction system) is set.PCR primer carries out 1.2% agarose gel electrophoresis detection.

Getting 3 pipe inoculations has the LB nutrient solution of mono-clonal bacterium colony to carry out bacterium liquid PCR positive clone identification, arranges negative control group simultaneously.The PCR primer clip size of result display 2, No. 3 clones is about 290bp, points out it to be pMSCVpuro-hCC16 Positive recombinant clones (Fig. 1).Extract the plasmid DNA of 2, No. 3 clones, warp xhoi and EcoRi double digestion, obtains 6176bp and 290bp two fragments (Fig. 2), consistent with expected results.

Embodiment 2

HEK 293T cell is 37 DEG C and 5%CO in the high sugar (10% new-born calf serum, penicillin 100u/ml, Streptomycin sulphate 100ug/ml) of substratum DMEM ₂grow under condition, within 3 ~ 4 days, change liquid once.

Cell transfecting adopts 6cm culture dish, when Growth of Cells to 80 ~ 90% merges, operates according to transfection reagent specification sheets, transfection 2 μ g pMSCVpuro-hCC16 plasmid, and the empty plasmid pMSCVpuro of the equivalent of transfection simultaneously in contrast; 48 h before harvest cells, RIPA lysate extracts protein, and Western blotting carries out the qualification of hCC16 expression.Namely BCA method is adopted to carry out quantitatively to protein extract, get 20 μ g total proteins and carry out 12% SDS-PAGE separation, albumen subsequently on gel is after transferring film, closed supervisor, respectively with rabbit anti-human CC16 antibody (1: 1000), the anti-Flag monoclonal antibody (1: 1000) of people for primary antibodie, goat antirabbit/people HRP-IgG (1: 5000) be two resist, adopt ECL luminescent solution, condense imager analytical results with BIO-RAD.Result shows, and have the expression (Fig. 3) of Clara cell 10kDa protein, and the cell of transfection empty plasmid has no expression in the cell of transfection Eukaryotic expression recombinant plasmid pMSCVpuro-hCC16.

Adopt the transfection of 15cm culture dish, every ware transfection 6 μ g plasmid DNA, transfection is after 24 hours, and adding final concentration is the screening that the tetracycline of 1.5 μ g/ml carries out stably express cell clone, and after 48 hours, survivaling cell is the stabilized cell clone containing pMSCVpuro-hCC16 plasmid.Be replaced by serum free medium, continue cultivation 48 hours, collect supernatant, for purification of Recombinant Clara cell 10kDa protein; Collecting cell, adds protein lysate (50mM Tris-HCl, pH7.4 simultaneously; 150mM NaCl; 1mM EDTA; 1%TRITON X-100), obtain solubility plasmosin, for purification of Recombinant Clara cell 10kDa protein.

Embodiment 3

Collect above-mentioned serum free medium.Remaining cell, obtains the cell of stable transfection pMSCVpuro-hCC16, adds 1.5 milliliters of protein lysates (50mM Tris-HCl, pH7.4 with PBS washing; 150mM NaCl; 1mM EDTA; 1%TRITON X-100), hatch 30min, use cell to scrape collecting cell lysate, the centrifugal 10min of 12000g, collect supernatant, be placed on ice.At 4 DEG C, the serum free medium of collection and cell pyrolysis liquid are added the ANTI-FLAG M using TBS (50mM Tris HCl, 150mM NaCl, pH7.4) to balance in advance ₂in affinity gel, make albumen and gel overnight incubation.Second day, wash unconjugated albumen off with the TBS of 20 times of column volumes.Add 6ml 0.1M glycine-HCl, pH3.5, the target protein of elution of bound.600 μ l 0.5M Tris HCl are added, pH7.4 in elutriant; 1.5M NaCl, uses the evaporating pipe that molecular weight cut-off is 3kDa to concentrate target protein, and adjustment protein concentration is 1 μ g/ μ l.By obtained albumen in-80 DEG C of preservations.

The people's Clara cell 10kDa protein getting 1 μ g purifying carries out 15% SDS-PAGE separation, detects purifying protein in conjunction with coomassie brilliant blue R_250, and result display obtains purity recombination fusion protein Flag-rCC16(Fig. 4 more than 99%).

Embodiment 4

Yelkin TTS is at PLA ₂and Ca ²⁺effect is lower generates free fatty acids and lysophospholipid, and reaction product is acid.By understanding the amount of acid product, calculate PLA ₂active.A PLA ₂activity unit (U) is defined as: under 37 DEG C of conditions, and the every mL sample reaction of per minute consumes 1nmolL ^-1hydrochloric acid (1U ≡ 1 nmol× mL ¹× min ¹).

For detecting the biologic activity of people's Clara cell 10kDa protein matter of purifying, the purifying protein of various dose is added following table and contains Yelkin TTS and PLA by the present embodiment ₂reaction system in, after reaction terminates, measure control tube and measure the pH value of pipe.The pH value of control tube is titrated to the dilute hydrochloric acid of 0.004M the pH value measuring pipe, records the dilute hydrochloric acid amount (ml) consumed.

The PLA of each sample ₂active U is calculated as follows: PLA ₂(U)=(0.004 × 10 ⁶× 2.5 × V)/T.

Wherein, 0.004 is the volumetric molar concentration of dilute hydrochloric acid, and control tube pH value is titrated to the hydrochloric acid volume (ml) consumed when measuring pipe pH value by V in this sample determination, T is reaction times (60min).

Experimental data adopts SPSS13.0 statistics software to process, calculating chart with mean ± standard deviation ( ± s) represent, compare application variance analysis between group, represent that difference has significance with P < 0.05.

Experimental result shows, PLA ₂activity along with the increase of recombinant human Clara cell 10kDa protein matter dosage obviously decline (Fig. 5).

Claims (7)

1. a recombinant human CC16 gene, has the nucleotide sequence shown in SEQ ID NO.1.

2. the Eukaryotic expression recombinant plasmid containing recombinant human CC16 gene described in claim 1, be with xhoi and ecoRrecombinant human CC16 gene and the carrier for expression of eukaryon pMSVpuro of I double digestion connect and compose, and have the nucleotide sequence shown in SEQ ID NO.2.

3. the eukaryotic cell expression system containing Eukaryotic expression recombinant plasmid described in claim 2 is proceeded in HEK 293T cell by described Eukaryotic expression recombinant plasmid to build.

4. the method for Restruction people Clara cell 10kDa protein in HEK 293T cell expression system, comprises the following steps:

1) to verify the nucleotide sequence of answering according to people's Clara cell 10kDa protein, synthesize recombinant human CC16 gene according to claim 1;

2) recombinant human CC16 gene is connected in carrier for expression of eukaryon pMSVpuro, builds the Eukaryotic expression recombinant plasmid pMSCVpuro-hCC16 containing described recombinant human CC16 gene;

3) with described Eukaryotic expression recombinant plasmid pMSCVpuro-hCC16 transfection HEK 293T cell, the HEK 293T stabilized cell expression system containing recombinant human CC16 gene is built;

4) in described HEK 293T stabilized cell expression system, culture expression recombinant human Clara cell 10kDa protein under serum free medium system;

5) also recombinant human Clara cell 10kDa protein described in purifying is separated;

Wherein, described step 2) in carrier for expression of eukaryon pMSCVpuro warp xhoi and ecoRconnect with the recombinant human CC16 gene of same double digestion after I double digestion.

5. the method for Restruction people Clara cell 10kDa protein according to claim 4, wherein said HEK 293T cell is containing 10% new-born calf serum, 100u/ml penicillin, in the DMEM high glucose medium of 100ug/ml Streptomycin sulphate, 37 DEG C and 5%CO ₂grow under condition, within 3 ~ 4 days, change liquid once.

6. the method for Restruction people Clara cell 10kDa protein according to claim 4, wherein said step 3) in, the tetracycline that final concentration is 1.5 μ g/ml is added after 24 hours, cell cultures 48 hours, to filter out the cell clone of stably express people Clara cell 10kDa protein in transfection.

7. the method for Restruction people Clara cell 10kDa protein according to claim 4, wherein step 5) separation of described recombinant human Clara cell 10kDa protein purge process be:

Collect step 4) serum free medium supernatant;

Remaining cell PBS washs, and obtains the cell of stable transfection pMSCVpuro-hCC16, hatches 30min with protein lysate, collecting cell lysate;

By centrifugal to cell pyrolysis liquid and the serum-free medium collected, collect supernatant, add at 4 DEG C in advance with the ANTI-FLAG M that TBS balances ₂in affinity gel, make albumen and gel overnight incubation;

Wash the non-associated proteins on affinity gel off with TBS after, then with the target protein that elution affinity gel combines, add 0.5M Tris HCl in elutriant, pH7.4; 1.5M NaCl, uses the evaporating pipe of molecular weight cut-off 3kDa to concentrate target protein, obtains the recombinant human Clara cell 10kDa protein of purifying;

Wherein, described protein lysate contains 50mM Tris-HCl, pH7.4; 150mM NaCl; 1mM EDTA; 1%TRITON X-100; Described TBS contains 50mM Tris HCl, 150mM NaCl, pH7.4; Described elutriant is 0.1M glycine-HCl, pH3.5.