本文選題：FHL2 + 原核表達(dá)�。� 參考：《南方醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 目的和意義 FHL2作為新確定的癌基因,其功能及作用機(jī)制尚不完全清楚。本研究通過構(gòu)建FHL2原核表達(dá)質(zhì)粒,在大腸桿菌中進(jìn)行表達(dá)并分離得到純化的FHL2重組蛋白,進(jìn)而制備FHL2抗血清,為FHL2的進(jìn)一步研究及臨床試劑盒開發(fā)提供實(shí)驗(yàn)材料。 材料和方法 1.主要材料 大腸腺癌細(xì)胞株Lovo,原核表達(dá)質(zhì)粒pET22b+,弗氏完全/不完全佐劑,新西蘭大白兔。 2.方法 2.1引物設(shè)計(jì) 上游引物AAC GAATTC C ATGACTGAGCGCTTTGACTG包含了EcoRI的酶切位點(diǎn),下游引物TTT GTCGAC GATGTCTTTCCCACAGTCGG包含了Sal I的酶切位點(diǎn)。用該對(duì)引物擴(kuò)增的PCR產(chǎn)物預(yù)期目的片段長(zhǎng)度為854bp。 2.2 FHL2基因原核表達(dá)質(zhì)粒的構(gòu)建 LOVO大腸癌細(xì)胞(由本室常規(guī)培養(yǎng)保存)用含有100 mL/L FCS的RMPI-1640培養(yǎng)液常規(guī)培養(yǎng)。用Trizol(TaKaRa公司產(chǎn)品)提取總RNA,逆轉(zhuǎn)錄反應(yīng)按試劑盒(寶曼靈公司產(chǎn)品)說明操作。取2μl cDNA用上游引物和下游引物進(jìn)行PCR反應(yīng)。反應(yīng)條件:50℃1h,94℃30s,55℃30s,72℃45s,6個(gè)循環(huán),94℃30s,65℃30s,72℃45s,32個(gè)循環(huán)。所得目的片段用限制性核酸內(nèi)切酶EcoRI、SalI雙酶切后插人pET22b+的EcoR I與Sal I之間,所得質(zhì)粒命名為pET2b+/FHL2,進(jìn)行測(cè)序。 2.3 FHL2基因在大腸桿菌中的表達(dá) 含有pET2b+/FHL2質(zhì)粒的大腸桿菌BL21(DE3)plysS接種含氨芐青霉素100μg/ml的LB培養(yǎng)基,震蕩培養(yǎng)8h,取0.5 ml過夜菌接種于含氨芐青霉素100μg/ml的LB培養(yǎng)液4.5 mL中,37℃劇烈震蕩培養(yǎng)4h,加入500 mmol/L IPTC至終濃度為1 mmol/L,25℃劇烈震蕩培養(yǎng)4h,取1ml菌液進(jìn)行SDS-PAGE鑒定。 2.4 rhFHL2蛋白的分離與純化 取1000mL誘導(dǎo)表達(dá)的菌液,10000轉(zhuǎn)/分4℃離心2min,菌體沉淀加入1/20細(xì)胞生長(zhǎng)體積的細(xì)菌裂解液和PMSF,超聲破碎細(xì)菌。10000轉(zhuǎn)/分,4℃離心15min。棄上清,往沉淀加入1/20原細(xì)菌生長(zhǎng)體積的UrNTA-0 Buffer(20mMTris-HCl pH7.9,0.5M NaCl,10%Glycerol,8M Urea)和PMSF,懸浮細(xì)菌,室溫放置30min。10000轉(zhuǎn)/分,4℃離心15min,取上清。用50ml的UrNTA-0 Buffer平衡5mLNTA層析柱。上清液上柱,收集穿透部分,用于SDS/PAGE分析蛋白質(zhì)的結(jié)合情況。用25mi的UrNTA-0 Buffer洗脫未結(jié)合成分,然后分別用25mlUrNTA-20,UrNTA-40,UrNTA-60,UrNTA-100,UrNTA-200,UrNTA-500(UrNTA-0Buffer含20-500mmol/L Midazole)洗脫,收集洗脫液,SDS-PAGE確定目標(biāo)蛋白質(zhì)在洗脫液中的分布情況。PBS透析24 h,每6h更換PBS 1次,BCA法測(cè)蛋白濃度,-70℃保存。 2.5兔抗重組融合蛋白rhFHL2抗血清的制備μ 取純化的融合蛋白rhFHL2 800 l(約800gg)與800 l弗氏完全佐劑徹底乳化,經(jīng)背部皮下多點(diǎn)注射新西蘭白兔。首次免疫后4周取純化的融合蛋白FHL2400 l(約400 g)與400 l弗氏不完全佐劑徹底乳化,進(jìn)行第2次免疫,之后第6,8周分別用rhFHL2加強(qiáng)免疫1次,最后一次免疫不加佐劑。最后1次加強(qiáng)免疫后8d耳緣靜脈采血2mL,測(cè)定血清中抗體的效價(jià)。并于最后1次免疫后10 d,頸動(dòng)脈插管放血,分離血清,置-80℃保存。 2.6 ELISA法測(cè)定效價(jià)及抗體的純化 純化后的融合蛋白rhFHL2按5 g/ml,50 l/孔,包被酶標(biāo)板,分別加入等比稀釋的血清,洗后加HRP標(biāo)記的羊抗兔IgG二抗孵育,最后加OPD底物液顯色,終止反應(yīng)后用酶標(biāo)儀測(cè)定OD492nm,以等于陰性對(duì)照孔平均值的2.1倍者判為陽性。 2.7兔抗融合蛋白rhFHL2抗體的純化 往10ml免疫兔血清中加等量的PBS(pH7.4);逐滴加入等量飽和硫酸銨溶液,于4℃靜置30 min;4℃4000轉(zhuǎn)/分離心15 min;棄上清,沉淀用20 ml預(yù)冷的PBS溶解,逐滴加入10mL飽和硫酸銨溶液,置4℃冰箱30min;4℃4000轉(zhuǎn)/分離心15 min,收集沉淀,沉淀用20ml預(yù)冷的PBS溶解,獲得純化的IgG抗體。PBS透析24h,每6h更換PBS 1次,后加等體積的甘油,再加入疊氮鈉至終濃度0.02%,分裝后-70℃保存。 2.8兔抗rhFHL2抗體的特性鑒定 Lovo細(xì)胞高表達(dá)FHL2,用制備的兔抗融合蛋白抗體對(duì)其作Western blot分析。收集細(xì)胞,抽提蛋白,蛋白樣品變性后以40 g每孔上樣,SDS-PAGE電泳分離,然后將蛋白電轉(zhuǎn)移至PVDF膜,以封閉液(50 mmol/L Tris-Buffered-Saline,pH7.5,含1%BSA和0.1%Tween 20)于37℃振蕩封閉2h,再加入以封閉液稀釋(1:500)的兔抗rhFHL2抗血清,于4℃孵育過夜,洗膜后加1:2500稀釋的HRP-羊抗兔IgG于37℃溫育1h,充分洗滌后以DAB顯色。 SW480也同樣高表達(dá)FHL2,將SW480接種在蓋玻片上,用3.7%多聚甲醛固定,5%BSA封閉,滴加按1:200稀釋的FHL2抗血清,37℃孵育1h,經(jīng)PBST沖洗后,加入生物素化抗兔二抗,37℃孵育20min,再次PBST洗滌后,滴加SABC試劑,孵育20min,PBST洗滌,DAB顯色,蘇森素復(fù)染細(xì)胞核,封片后鏡下觀察。 結(jié)果 1、FHL2原核表達(dá)質(zhì)粒的構(gòu)建 通過RT-PCR獲得了854bp的FHL2基因片段,成功地構(gòu)建了FHL2的原核表達(dá)質(zhì)粒pET22b+/FHL2,經(jīng)測(cè)序與GenBank公布的一致。 2、融合蛋白的誘導(dǎo)表達(dá) 經(jīng)IPTG誘導(dǎo)后,含重組質(zhì)粒的大腸桿菌能夠表達(dá)相對(duì)分子質(zhì)量M:37084的重組rhFHL2蛋白,并且rhFHL2主要存在于沉淀部分,即rhFHL2是以不可溶性的包涵體形式存在。 3、抗體的制備純化 用純化的融合蛋白HIS-FHL2作抗原,免疫大白兔,所得的多克隆抗血清,用EL1SA檢測(cè)。抗血清的效價(jià)為1:12500。 4、抗人FHL2多克隆抗體的評(píng)價(jià) 用制備的多克隆抗體對(duì)融合蛋白HIS-FHL2作Western blot分析,在約40 KD處呈現(xiàn)特異性的結(jié)合條帶,表明所制備的抗體特異性極佳,用Western Blot分析時(shí)最低稀釋度為1:500,說明靈敏度極高。 免疫細(xì)胞實(shí)驗(yàn)和免疫組織實(shí)驗(yàn)也證實(shí)了抗FHL2可用應(yīng)于免疫細(xì)胞實(shí)驗(yàn)及免疫組織化學(xué)實(shí)驗(yàn),實(shí)驗(yàn)發(fā)現(xiàn)FHL2主要在細(xì)胞核中表達(dá),但細(xì)胞質(zhì)中有也少量表達(dá)。 結(jié)論 本研究中通過分子克隆技術(shù),在原核表達(dá)系統(tǒng)中,成功重組表達(dá)了FHL2,利用分離純化后的重組蛋白rhFHL2免疫新西蘭大白兔,成功獲得效價(jià)為1:12500(ELISA法)的抗FHL2的多克隆抗體血清,該抗體特異性強(qiáng),用western blotting檢測(cè)只有一條特異性條帶。后續(xù)實(shí)驗(yàn)中我們發(fā)現(xiàn)該抗體還可應(yīng)用于ELISA,免疫熒光,免疫共沉淀,免疫組化,并且效果良好,說明該抗體有廣泛的適用范圍。故本實(shí)驗(yàn)為進(jìn)一步深入研究FHL2蛋白的生物學(xué)功能及其在腫瘤免疫治療中的作用提供了方便快捷的檢測(cè)用抗體。
[Abstract]:Purpose and significance
As a newly identified oncogene, the function and mechanism of the FHL2 are not completely clear. By constructing the FHL2 prokaryotic expression plasmid, the recombinant protein of the purified FHL2 was expressed and isolated in Escherichia coli, and then the FHL2 antiserum was prepared, which provided the experimental materials for the further study of FHL2 and the development of the bed reagent kit.
Materials and methods
1. main materials
Colorectal adenocarcinoma cell line Lovo, prokaryotic expression plasmid pET22b+, Freund's complete / incomplete adjuvant, New Zealand white rabbit.
2. method
2.1 primer design
The upstream primer AAC GAATTC C ATGACTGAGCGCTTTGACTG contains the enzyme cut site of EcoRI, and the downstream primer TTT GTCGAC GATGTCTTTCCCACAGTCGG contains the enzyme tangent site of the Sal I. The PCR product amplified by the primers is expected to be the length of 854bp..
Construction of the prokaryotic expression plasmid of 2.2 FHL2 gene
LOVO colorectal cancer cells (conventional culture preserved in this room) were cultured in RMPI-1640 medium containing 100 mL/L FCS. The total RNA was extracted with Trizol (TaKaRa company product). The reverse transcriptase reaction was operated according to the reagent box (the product of the company). The upstream primers and lower primers were used for the reaction of PCR. The reaction conditions: 50 C 1H, 94 C 30s, 55 30s, 72 centigrade 45s, 6 cycles, 94 C 30s, 65 C 30s, 72 centigrade 45s, 32 cycles. The obtained target fragment is between SalI double enzyme and SalI double enzyme between EcoR I and Sal I. The obtained plasmid is named and sequenced.
Expression of 2.3 FHL2 gene in Escherichia coli
The Escherichia coli BL21 (DE3) plysS containing the pET2b+/FHL2 plasmid was inoculated with the LB medium containing ampicillin 100 u g/ml, concussion and culture 8h, and inoculated 0.5 ml overnight bacteria to LB culture liquid containing ampicillin 100 micron LB culture liquid 4.5 mL, 37 degrees centigrade intense concussion, 1 The liquid was identified by SDS-PAGE.
Separation and purification of 2.4 rhFHL2 protein
1000mL induced expression of bacterial liquid, 10000 turn / 4 centigrade centrifugation for 2min, bacteria precipitated into 1/20 cell growth volume of bacterial lysate and PMSF, ultrasonic breakup bacteria.10000 conversion / fraction, 4 C centrifuge 15min. abandoned supernatant, UrNTA-0 Buffer (20mMTris-HCl pH7.9,0.5M NaCl, 20mMTris-HCl, pH7.9,0.5M NaCl) to precipitate 1/20 probacteria. Suspension bacteria, room temperature 30min.10000 turn / sub, 4 C centrifuge 15min, take the supernatant. Use UrNTA-0 Buffer of 50ml to balance 5mLNTA column. The upper column of the supernatant is used for SDS/PAGE analysis of protein binding. The unbound components are eluted with 25mi UrNTA-0 Buffer, then 25mlUrNTA-20, UrNTA-40, UrNTA-40, societies, respectively, are used respectively. RNTA-200, UrNTA-500 (UrNTA-0Buffer containing 20-500mmol/L Midazole) elution, collection of eluent, SDS-PAGE determination of the distribution of target protein in the eluant.PBS dialysis 24 h, 1 times per 6h replacement PBS, BCA method of protein concentration, -70 C preservation.
Preparation of 2.5 Rabbit anti recombinant fusion protein rhFHL2 antiserum
The purified fusion protein rhFHL2 800 l (about 800gg) and 800 l Freund complete adjuvant were thoroughly emulsified. New Zealand white rabbits were injected subcutaneously into the back subcutaneous. After the first immunization, the purified fusion protein FHL2400 L (about 400 g) and 400 l Freund incomplete adjuvant were thoroughly emulsified, and second immunizations were carried out, and 1 times were strengthened with rhFHL2, respectively, at week 6,8, respectively. The last immunization was done without an adjuvant. The last 1 times to strengthen the 8D ear vein blood collection after immunization was 2mL, and the serum antibody titer was measured. After the last 1 times of immunization, 10 d, the carotid artery catheterization was blood, the serum was separated and stored at -80 C.
Determination of titer and antibody by 2.6 ELISA method
The purified fusion protein rhFHL2, according to the 5 g/ml, 50 l/ hole, and the enzyme labeled plate, was added to the same dilution serum respectively. After washing, the sheep with HRP labeled anti rabbit IgG two were incubated, and the OPD substrate liquid was added to the color, and the OD492nm was determined by the enzyme labeling instrument after the termination of the reaction, which was equal to the positive of the 2.1 times of the negative control Kong Ping mean.
Purification of 2.7 Rabbit anti fusion protein rhFHL2 antibody
Adding equal amount of PBS (pH7.4) to 10ml immunized rabbit serum, adding an equal amount of saturated ammonium sulfate by drop by drop, 30 min at 4 C, 4000 turn at 4 C / 15 min, discarding the supernatant, settling with 20 ml precooled PBS, adding 10mL saturated ammonium sulphate solution by drop by drop to 4 C refrigerators 30min, 4 C 4000 / 15 min, collecting precipitation and 20ml precooling for precipitation. PBS was dissolved, the purified IgG antibody.PBS was dialytic 24h, PBS 1 times per 6h, then equal volume of glycerol, and then sodium azide to the final concentration of 0.02%, then -70 C was stored.
Identification of anti rhFHL2 antibody in 2.8 rabbits
Lovo cells expressed high expression of FHL2, using the Rabbit anti fusion protein antibody prepared by the prepared rabbit for Western blot analysis. Collect cells, extract protein, protein samples denatured with 40 g per pore sample and SDS-PAGE electrophoresis, then transfer the protein to PVDF membrane and oscillate at 37 centigrade with closed liquid (50 mmol/L Tris-Buffered-Saline, pH7.5, 1%BSA and 0.1%Tween 20). 2H was closed, then the Rabbit anti rhFHL2 antiserum was added with closed liquid dilution (1:500), and incubated at 4 C for night. After washing the film and adding 1:2500 diluted HRP- sheep, the rabbit IgG was incubated at 37 C for 1h, and after full washing, the color was displayed with DAB.
SW480 also highly expressed FHL2, inoculating SW480 on the cover glass, immobilized with 3.7% polyformaldehyde, 5%BSA closed, FHL2 antiserum diluted by 1:200, incubating 1H at 37 degrees C, adding biotinylated anti rabbit two resistance and incubating 20min at 37 C after PBST rinse, and then adding SABC reagent, incubating 20min, showing color and SUSON redyeing The nucleus and the seal were observed under the microscope.
Result
1, construction of the prokaryotic expression plasmid of FHL2
The FHL2 gene fragment of 854bp was obtained by RT-PCR, and the prokaryotic expression plasmid pET22b+/FHL2 of FHL2 was successfully constructed, which was consistent with GenBank published by sequencing.
2, the induced expression of fusion protein
After IPTG induction, the recombinant plasmid containing Escherichia coli can express the recombinant rhFHL2 protein of relative molecular mass M:37084, and rhFHL2 mainly exists in the precipitate part, that is, rhFHL2 is in the form of insoluble inclusion body.
3, preparation and purification of antibody
Using purified fusion protein HIS-FHL2 as antigen, the polyclonal antisera obtained from immunized rabbits were detected by EL1SA. The titer of antiserum was 1:12500.
4, evaluation of anti human FHL2 polyclonal antibody
Using the polyclonal antibody prepared by the polyclonal antibody to the Western blot analysis of the fusion protein HIS-FHL2, the specific binding band was presented at about 40 KD, indicating that the antibody specificity was excellent. The lowest dilution degree was 1:500 with Western Blot analysis, indicating high sensitivity.
Immune cell experiments and immuno tissue experiments also confirmed that anti FHL2 should be used in immuno cell experiments and immunohistochemistry experiments. The experiment found that FHL2 is mainly expressed in the nucleus, but a small amount of expression in the cytoplasm is also found.
conclusion
In this study, FHL2 was successfully expressed in the prokaryotic expression system by molecular cloning technology. The recombinant protein rhFHL2 of New Zealand white rabbit was immunized with the purified recombinant protein, and the anti FHL2 polyclonal antibody sera of 1:12500 (ELISA method) was successfully obtained. The antibody was highly specific and only one specific strip was detected by Western blotting. In the follow-up experiment, we found that the antibody can also be applied to ELISA, immunofluorescence, immunofluorescence, immunohistochemistry, and the effect is good, indicating that the antibody has a wide range of application. Therefore, this experiment provides a convenient and quick detection for the further in-depth study of the biological function of FHL2 protein and its role in the treatment of cancer free disease. Antibodies.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R735;R392

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