重组FHL2的原核表达、纯化及抗血清的制备

发布时间：2018-06-20 01:05

本文选题：FHL2 + 原核表达　；参考：《南方医科大学》2008年硕士论文

【摘要】： 目的和意义 FHL2作为新确定的癌基因,其功能及作用机制尚不完全清楚。本研究通过构建FHL2原核表达质粒,在大肠杆菌中进行表达并分离得到纯化的FHL2重组蛋白,进而制备FHL2抗血清,为FHL2的进一步研究及临床试剂盒开发提供实验材料。材料和方法 1.主要材料大肠腺癌细胞株Lovo,原核表达质粒pET22b+,弗氏完全/不完全佐剂,新西兰大白兔。 2.方法 2.1引物设计上游引物AAC GAATTC C ATGACTGAGCGCTTTGACTG包含了EcoRI的酶切位点,下游引物TTT GTCGAC GATGTCTTTCCCACAGTCGG包含了Sal I的酶切位点。用该对引物扩增的PCR产物预期目的片段长度为854bp。 2.2 FHL2基因原核表达质粒的构建 LOVO大肠癌细胞(由本室常规培养保存)用含有100 mL/L FCS的RMPI-1640培养液常规培养。用Trizol(TaKaRa公司产品)提取总RNA,逆转录反应按试剂盒(宝曼灵公司产品)说明操作。取2μl cDNA用上游引物和下游引物进行PCR反应。反应条件:50℃1h,94℃30s,55℃30s,72℃45s,6个循环,94℃30s,65℃30s,72℃45s,32个循环。所得目的片段用限制性核酸内切酶EcoRI、SalI双酶切后插人pET22b+的EcoR I与Sal I之间,所得质粒命名为pET2b+/FHL2,进行测序。 2.3 FHL2基因在大肠杆菌中的表达含有pET2b+/FHL2质粒的大肠杆菌BL21(DE3)plysS接种含氨苄青霉素100μg/ml的LB培养基,震荡培养8h,取0.5 ml过夜菌接种于含氨苄青霉素100μg/ml的LB培养液4.5 mL中,37℃剧烈震荡培养4h,加入500 mmol/L IPTC至终浓度为1 mmol/L,25℃剧烈震荡培养4h,取1ml菌液进行SDS-PAGE鉴定。 2.4 rhFHL2蛋白的分离与纯化取1000mL诱导表达的菌液,10000转/分4℃离心2min,菌体沉淀加入1/20细胞生长体积的细菌裂解液和PMSF,超声破碎细菌。10000转/分,4℃离心15min。弃上清,往沉淀加入1/20原细菌生长体积的UrNTA-0 Buffer(20mMTris-HCl pH7.9,0.5M NaCl,10%Glycerol,8M Urea)和PMSF,悬浮细菌,室温放置30min。10000转/分,4℃离心15min,取上清。用50ml的UrNTA-0 Buffer平衡5mLNTA层析柱。上清液上柱,收集穿透部分,用于SDS/PAGE分析蛋白质的结合情况。用25mi的UrNTA-0 Buffer洗脱未结合成分,然后分别用25mlUrNTA-20,UrNTA-40,UrNTA-60,UrNTA-100,UrNTA-200,UrNTA-500(UrNTA-0Buffer含20-500mmol/L Midazole)洗脱,收集洗脱液,SDS-PAGE确定目标蛋白质在洗脱液中的分布情况。PBS透析24 h,每6h更换PBS 1次,BCA法测蛋白浓度,-70℃保存。 2.5兔抗重组融合蛋白rhFHL2抗血清的制备μ 取纯化的融合蛋白rhFHL2 800 l(约800gg)与800 l弗氏完全佐剂彻底乳化,经背部皮下多点注射新西兰白兔。首次免疫后4周取纯化的融合蛋白FHL2400 l(约400 g)与400 l弗氏不完全佐剂彻底乳化,进行第2次免疫,之后第6,8周分别用rhFHL2加强免疫1次,最后一次免疫不加佐剂。最后1次加强免疫后8d耳缘静脉采血2mL,测定血清中抗体的效价。并于最后1次免疫后10 d,颈动脉插管放血,分离血清,置-80℃保存。 2.6 ELISA法测定效价及抗体的纯化纯化后的融合蛋白rhFHL2按5 g/ml,50 l/孔,包被酶标板,分别加入等比稀释的血清,洗后加HRP标记的羊抗兔IgG二抗孵育,最后加OPD底物液显色,终止反应后用酶标仪测定OD492nm,以等于阴性对照孔平均值的2.1倍者判为阳性。 2.7兔抗融合蛋白rhFHL2抗体的纯化往10ml免疫兔血清中加等量的PBS(pH7.4);逐滴加入等量饱和硫酸铵溶液,于4℃静置30 min;4℃4000转/分离心15 min;弃上清,沉淀用20 ml预冷的PBS溶解,逐滴加入10mL饱和硫酸铵溶液,置4℃冰箱30min;4℃4000转/分离心15 min,收集沉淀,沉淀用20ml预冷的PBS溶解,获得纯化的IgG抗体。PBS透析24h,每6h更换PBS 1次,后加等体积的甘油,再加入叠氮钠至终浓度0.02%,分装后-70℃保存。 2.8兔抗rhFHL2抗体的特性鉴定 Lovo细胞高表达FHL2,用制备的兔抗融合蛋白抗体对其作Western blot分析。收集细胞,抽提蛋白,蛋白样品变性后以40 g每孔上样,SDS-PAGE电泳分离,然后将蛋白电转移至PVDF膜,以封闭液(50 mmol/L Tris-Buffered-Saline,pH7.5,含1%BSA和0.1%Tween 20)于37℃振荡封闭2h,再加入以封闭液稀释(1:500)的兔抗rhFHL2抗血清,于4℃孵育过夜,洗膜后加1:2500稀释的HRP-羊抗兔IgG于37℃温育1h,充分洗涤后以DAB显色。 SW480也同样高表达FHL2,将SW480接种在盖玻片上,用3.7%多聚甲醛固定,5%BSA封闭,滴加按1:200稀释的FHL2抗血清,37℃孵育1h,经PBST冲洗后,加入生物素化抗兔二抗,37℃孵育20min,再次PBST洗涤后,滴加SABC试剂,孵育20min,PBST洗涤,DAB显色,苏森素复染细胞核,封片后镜下观察。结果 1、FHL2原核表达质粒的构建通过RT-PCR获得了854bp的FHL2基因片段,成功地构建了FHL2的原核表达质粒pET22b+/FHL2,经测序与GenBank公布的一致。 2、融合蛋白的诱导表达经IPTG诱导后,含重组质粒的大肠杆菌能够表达相对分子质量M:37084的重组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体形式存在。 3、抗体的制备纯化用纯化的融合蛋白HIS-FHL2作抗原,免疫大白兔,所得的多克隆抗血清,用EL1SA检测。抗血清的效价为1:12500。 4、抗人FHL2多克隆抗体的评价用制备的多克隆抗体对融合蛋白HIS-FHL2作Western blot分析,在约40 KD处呈现特异性的结合条带,表明所制备的抗体特异性极佳,用Western Blot分析时最低稀释度为1:500,说明灵敏度极高。免疫细胞实验和免疫组织实验也证实了抗FHL2可用应于免疫细胞实验及免疫组织化学实验,实验发现FHL2主要在细胞核中表达,但细胞质中有也少量表达。结论本研究中通过分子克隆技术,在原核表达系统中,成功重组表达了FHL2,利用分离纯化后的重组蛋白rhFHL2免疫新西兰大白兔,成功获得效价为1:12500(ELISA法)的抗FHL2的多克隆抗体血清,该抗体特异性强,用western blotting检测只有一条特异性条带。后续实验中我们发现该抗体还可应用于ELISA,免疫荧光,免疫共沉淀,免疫组化,并且效果良好,说明该抗体有广泛的适用范围。故本实验为进一步深入研究FHL2蛋白的生物学功能及其在肿瘤免疫治疗中的作用提供了方便快捷的检测用抗体。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R735;R392

【共引文献】