肝螺杆菌甲基基团接受趋化信号转导蛋白多克隆抗体的制备及鉴定

发布时间：2018-05-31 04:42

本文选题：胆型螺杆菌 + 分离　；参考：《南方医科大学》2010年硕士论文

【摘要】： 目的和意义肝螺杆菌(Helicobacter hepaticus, Hh)致病机制及流行病学特征尚不完全清楚。本研究通过制备肝螺杆菌甲基基团接受趋化信号转导蛋白(methyl-accepting chemotaxis signal transduction protein, MCP)兔抗血清,为进一步探讨肝螺杆菌在实验动物及人类的感染及及临床检测试剂盒的开发提供实验基础。材料和方法 1、主要材料：细菌菌株(肝螺杆菌、幽门螺杆菌),空肠弯曲菌琼脂基础,弗氏完全/不完全佐剂,辣根过氧化物酶标记山羊抗兔IgG。Balb/C小鼠,新西兰大白兔。 2、方法 2.1细菌分离及培养 2.1.1胆型螺杆菌的分离及鉴定参照文献方法分离胆型螺杆菌。SPF (special pathogene free)级Balb/C小鼠禁食48h后,取肠道组织匀浆,涂布于空肠弯曲菌选择性血琼脂培养基(含7%冻融脱纤维羊血,选择性抗生素万古霉素10mg/L,多粘菌素B 2500单位/L,二性霉素B 10mg/L, TMP 5mg/L),37℃微需氧条件(5%O2.10%CO2及85%N2)下培养3-5天,每天观察有无菌落生长。挑取可疑菌落传代培养。细菌鉴定：1、形态鉴定可疑细菌涂片革兰染色,油镜下观察细菌形态。2、生化鉴定可疑细菌行快速尿素酶、氧化酶、过氧化物酶及硝酸还原酶实验(检测试剂盒由环凯生化公司提供)3、PCR扩增细菌16srRNA基因将透明、针尖大小的菌落用棉签刮至EP管中,PBS冲洗三次后,用细菌基因组DNA提取试剂盒提取细菌DNA。以可疑菌基因组DNA为模板,采用螺杆菌属特异性引物行PCR扩增。反应条件为94℃10min总变性,94℃30s,54℃30s,72℃55s,共35个循环,72℃10min总延伸。扩增产物于1.5%琼脂糖凝胶中电泳,电泳条件80V,30min。阳性结果测序分析(测序工作由英俊生物公司完成)。 2.1.2细菌传代培养取肝螺杆菌、胆型螺杆菌、幽门螺杆菌保种液各100μl涂布于空肠弯曲菌选择性血琼脂培养基,37℃微需氧条件下培养3天,细菌涂片革兰染色观察细菌生长情况。 2.2提取细菌外膜蛋白(cell surface proteins,CSPs) 细菌在空肠弯曲菌选择性血琼脂培养基培养(培养条件同前)48-72h后,用灭菌双蒸水洗刮下菌苔,室温4000 r/min,20 min离心洗涤菌液。重复洗涤一次。弃上清,收集菌泥,称重,按每4g菌泥加入0.2mol /L、pH 2.2甘氨酸-盐酸缓冲液100 ml,4℃磁力搅拌30 min,后在4℃条件下12000 r/min,离心15 min。透析,冷冻干燥法浓缩。BCA法测蛋白浓度。 2.3 MCP基因在大肠杆菌中的表达将我室保种的重组质粒pET22b+/MCP转化大肠杆菌感受态BL21(DE3),挑单克隆接种于10ml含氨苄青霉素100μg/ml的LB培养基,37℃200rpm培养3个半小时至OD600约为0.6-1.0。转接100μl于10ml含氨苄青霉素100μg/ml的LB培养基中,37℃200rpm培养1.5小时至OD600约为0.6,加入500mmol/L IPTG至终浓度为1mmol/L,37℃180rpm震荡培养4小时。将诱导菌液12000rpm 4℃离心5min,取1ml菌液沉淀进行SDS-PAGE鉴定。重组蛋白rhMCP包含表达载体pET22b(+)中的6×His标签,因此用Western blot对rhMCP的表达再次进行鉴定。 2.4 rhMCP蛋白的纯化取1000ml诱导表达的菌液,12000rpm 4℃离心5min,菌体沉淀加入1/20细菌生长体积的细菌裂解液和PMSF,冰上超声破碎细菌,10000rpm 4℃离心15min。弃上清,往沉淀中加入1/20细菌生长体积的UrNTA-0 Buffer和PMSF,将细胞悬浮,室温放置30min, 10000rpm 4℃离心15min,取上清。用30ml的UrNTA-0 Buffer平衡3ml NTA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。15ml UrNTA-0 Buffer洗脱未结合部分,然后分别用15mlUrNTA-20, UrNTA-40, UrNTA-60, UrNTA-100, UrNTA-200, UrNTA-500洗脱,收集洗脱液,用SDS-PAGE确定目标蛋白质在洗脱液中的分布情况。用梯度PBS-尿素4℃透析(6M-4M-2M-1M),每4小时换液一次,最后用PBS 4℃透析24小时。BCA法测蛋白浓度,-70℃保存。 2.5兔抗重组融合蛋白MCP抗血清的制备取MCP重组蛋白800μl(约800μg)与800μl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MCP 400μl(约400μg)与400μl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MCP加上等体积弗氏不完全佐剂加强免疫,最后一次免疫不加佐剂。最后1次加强免疫后5-7天耳缘静脉采血2mL,行Western blot检测血清中是否有特异性抗体,并用间接ELISA法测定血清中抗体的效价。检测到特异性抗体后颈动脉插管放血,分离血清,置-80℃保存。 2.6 ELISA法测定效价纯化的融合蛋白MCP按5μg/ml,50μl/孔,包被酶标板,分别加人不同倍数稀释的血清,PBST冲洗后加HRP标记的羊抗兔IgG二抗孵育,最后加TMB底物液显色,终止后用酶标仪测定OD450nm,以等于阴性对照孔的2.1倍者判为阳性。 2.7兔抗融合蛋白MCP抗体的纯化 2.7.1饱和硫酸铵法初纯抗体往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30min; 4℃2000rpm/min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; 2000rpm离心15min,收集沉淀,沉淀用20ml预冷的PBS溶解,获得初步纯化的IgG抗体溶液。 2.7.2抗原特异性纯化法纯化抗体先用PH8.3的碳酸盐缓冲液预平衡CNBr-activated Sepharose 4B层析柱,加入纯化的MCP蛋白5mg,4℃过夜。次日离心去除未结合的蛋白,封闭非特异结合位点后加入粗纯抗体5mL于4℃摇床过夜。向柱中加入洗脱液离心后,吸取上清即为能与重组蛋白MCP特异结合的兔抗MCP抗体。加入等体积的甘油及叠氮钠至终浓度0.02%,分装后-70℃保存。 2.8兔抗MCP抗体的特性鉴定 Western blot检测抗体特异性取MCP重组蛋白、肝螺杆菌、幽门螺杆菌、及胆型螺杆菌菌体外膜蛋白CSPs变性后行SDS-PAG凝胶电泳,电转移至PVDF膜,以封闭液(50 mmol/L Tris-Buffered-Saline, pH7.5,含1%BSA和0.1% Tween20)于37℃振荡封闭2h,再加入以封闭液稀释(1：500)的兔抗MCP纯化抗体,于4℃孵育过夜,洗膜后加1：5000稀释的HRP-羊抗兔IgG(武汉博士德公司产品)于37℃温育1h,充分洗涤后ECL发光,显影。结果 1、胆型螺杆菌的分离及鉴定可疑细菌在空肠弯曲菌选择性血平板上微需氧培养3天后,肉眼可见针尖大小、雾状透明菌落,细菌涂片革兰染色(图3-1),见细菌革兰染色阴性,呈“S”或“C”形,与幽门螺杆菌相似,但菌体更细长,螺旋也较多。生化鉴定示细菌尿素酶实验阳性,氧化酶、过氧化物酶实验阳性,硝酸还原酶阴性。采用螺杆菌属特异性引物及胆型螺杆菌特异性引物扩增16s rRNA,1.5%琼脂糖凝胶电泳,于780b有阳性条带(图3-2,图3-3),产物测序结果与GeneBank库中基因组同源性对比,结果与Helicobacter bilis ATCC43879同源性达到99%,证实本实验所分离的细菌即为胆型螺杆菌(图3-4)。肝螺杆菌、胆型螺杆菌及幽门螺杆菌在空肠弯曲菌血平板上培养3天后,肉眼可见针尖大小、雾状透明菌落。 2、细菌菌体外膜蛋白CSPs的制备制备的细菌外膜蛋白经过浓缩后行SDS-PAGE电泳,考马斯亮蓝染色结果如图3-5所示。 3、重组融合蛋白的诱导表达经IPTG诱导后,含重组质粒的大肠杆菌E.coli BL21(DE3)能够表达相对分子质量M:14118D的重组蛋白rhMCP,前期试验鉴定rhMCP是以不可溶性的包涵体形式存在。经Western blot鉴定,在约14 KD处可见一特异性条带。 4、重组蛋白的分离纯化由于诱导表达的rhMCP是以不可溶的包涵体形式存在,因此用8M尿素溶解包涵体,再按变性蛋白纯化方法进行梯度尿素洗脱,其中以UrNTA200(咪唑浓度为200mM)洗脱效果最佳,纯化后的rhMCP用透析袋进行梯度尿素-PBS透析复性,复性后得到可溶性rhMCP,用BCA法测蛋白浓度为2.08mg/ml。 5、兔抗MCP多克隆抗体的制备纯化用纯化的融合蛋白His-MCP作抗原,免疫大白兔,所得的多克隆抗血清,行Western blotting及EL1SA检测(图3-6,图3-7),可见14KD处有特异性条带,抗血清的效价为1：32000,证明该实验制备成功高特异性、高效价的兔抗MCP多克隆抗体。 6、Western blotting进一步分析抗体特异性用制备的细菌外膜蛋白CSPs及MCP行SDS-PAGE电泳后,转移至PVDF膜上,依次加入制备的一抗及羊抗兔二抗后,ECL发光,胶片感光、显影,见肝螺杆菌及MCP蛋白在14KD处可见特异性条带,而胆型螺杆菌及幽门螺杆菌无条带,表明所制备的抗体具有较好的活性,可与免疫原特异性结合(图3-8)。结论本研究中成功从Balb/c小鼠结肠中分离出胆型螺杆菌。利用分离纯化后的重组蛋白MCP免疫新西兰大白兔,成功获得抗MCP的多克隆抗体血清,该抗体特异性强,纯化后用Western blotting检测只有一条特异性条带,且效价为1：32000(ELISA法)。再后来的实验中我们发现该抗体用于Western blotting检测细菌外膜蛋白CSPs,只有肝螺杆菌组有阳性条带,说明该抗体与其他常见螺杆菌属无交叉反应。故本实验为进一步深入研究肝螺杆菌的流行病学及制备简便的检测试剂盒提供了重要的实验基础。
[Abstract]:Purpose and significance
The pathogenic mechanism and epidemiological characteristics of Helicobacter hepaticus (Hh) are not completely clear. This study was made by preparing the methyl group of Helicobacter pylori to accept chemotaxis protein (methyl-accepting chemotaxis signal transduction protein, MCP) rabbit antiserum, in order to further explore the presence of Helicobacter in experimental animals and human beings. The development of infection and clinical detection kit provides experimental basis.
Materials and methods
1, the main materials: bacterial strains (Helicobacter pylori, Helicobacter pylori), agar base of Campylobacter jejuni, Freund complete / incomplete adjuvant, horseradish peroxidase labeled Goat anti rabbit IgG.Balb/C mice, New Zealand white rabbit.
2, method
2.1 isolation and culture of bacteria
Isolation and identification of 2.1.1 Helicobacter pylori
.SPF (special pathogene free) Balb/C mice were isolated from 48h by reference methods. The intestinal tissue homogenate was extracted from the intestinal tissue homogenate and coated on the selective blood agar medium of Campylobacter jejuni (including 7% freeze-thaw defibrinous sheep blood, selective antibiotic vancomycin 10mg/L, polymyxin B 2500 unit /L, amphotericin B 10mg/L, TMP), 37 degrees C Microaerobic conditions (5%O2.10%CO2 and 85%N2) were cultured for 3-5 days, and sterile growth was observed every day. Suspicious colonies were picked up for subculture.
Identification of bacteria: 1, morphological identification of bacteria smear gram blue staining, oil microscopic observation of bacterial morphology.2, biochemical identification of suspicious bacteria for rapid urease, oxidase, peroxidase and nitrate reductase experiment (detection kit provided by CKC) 3, PCR amplification bacterial 16srRNA gene will be transparent, needle tip size of the colony with cotton swab scratch In the EP tube, after three times of PBS flushing, bacterial genomic DNA extraction kit was used to extract bacterial DNA. from the genomic DNA of suspected bacteria as a template, and PCR was amplified by the specific primers of Helicobacter. The reaction conditions were 94 C 10min total denaturation, 94 C 30s, 54 C, 72 C 55s, 72 C 10min total extension. The amplified product was in 1.5% agarose gel. Electrophoresis, electrophoresis conditions 80V, 30min. positive results sequencing analysis (sequencing work completed by handsome biological company).
2.1.2 bacterial culture
Helicobacter pylori, Helicobacter pylori and Helicobacter pylori were coated on the selective blood agar medium of Campylobacter jejuni, and cultured for 3 days under microaerobic conditions at 37 degrees, and bacterial growth was observed by bacterial smear Gram staining.
2.2 extract cell surface proteins (CSPs).
After the bacteria were cultured in the selective blood agar medium of Campylobacter jejuni (pre culture condition) 48-72h, the bacteria were washed and scraped with double steamed water, at room temperature 4000 r/min and 20 min centrifuge washing bacteria. Once repeated washing, abandoned supernatant, collecting bacteria mud, weighing, adding 0.2mol /L to every 4G mud, pH 2.2 glycine hydrochloric acid buffer solution 100 ml, 4 C magnetic stirring 30 Min, after 12000 r/min at 4 C, centrifugation 15 min. dialysis, freeze drying concentration.BCA method to determine protein concentration.
Expression of 2.3 MCP gene in Escherichia coli
The recombinant plasmid pET22b+/MCP of the species in our room was transformed into Escherichia coli receptive BL21 (DE3) and selected to be inoculated to LB medium containing ampicillin 100 u g/ml in 10ml. At 37, 200rpm was cultured for 3.5 hours to OD600 about 0.6-1.0. transfer to 100 mu L in 10ml containing ampicillin 100 micron g/ml. 1.5 hours were cultured for 1.5 hours. For 0.6, 500mmol/L IPTG was added to the final concentration of 1mmol/L and 37 180rpm for 4 hours. The induced bacterial solution was centrifuged at 12000rpm 4 C, and 1ml bacteria solution was precipitated for SDS-PAGE identification. The recombinant protein rhMCP contained 6 x His labels in the expression vector pET22b (+), so the expression was identified again with Western.
Purification of 2.4 rhMCP protein
The bacteria solution induced by 1000ml was centrifuged for 5min at 12000rpm 4 C, the bacteria were precipitated into the bacterial lysate and PMSF of the growth volume of the 1/20 bacteria, the ice ultrasonic broken bacteria and 10000rpm at 4 C were centrifuged and 15min. abandoned the supernatant, and the UrNTA-0 Buffer and PMSF of the growth volume of 1/20 bacteria were added to the precipitation, and the cells were suspended and placed 30min at room temperature at 4 degrees. The heart 15min, take the supernatant. Balance the 3ml NTA column with the UrNTA-0 Buffer of 30ml, the upper column of the supernatant, and collect the penetrating part. It is used for the SDS-PAGE analysis of the binding of protein,.15ml UrNTA-0 Buffer eluting the unbound part, and then using 15mlUrNTA-20, UrNTA-40, desorption, desorption, and eluant collection, respectively. GE determined the distribution of the target protein in the eluent. Using gradient PBS- urea at 4 centigrade dialysis (6M-4M-2M-1M), the liquid was changed every 4 hours, and the protein concentration was measured at PBS 4 C for 24 hours.BCA, and stored at -70 C.
Preparation of antiserum against recombinant fusion protein MCP MCP in rabbits
MCP recombinant protein 800 Mu L (about 800 g) and 800 u l Freund complete adjuvant were thoroughly emulsified. New Zealand white rabbits were injected subcutaneously subcutaneously on the back. 4 weeks after the first immunization, the purified fusion protein MCP 400 micron L (about 400 mu g) and 400 u l Freund incomplete adjuvant were thoroughly emulsified, and second times of immunization were carried out, and then MCP plus the upper equal volume Freund was not finished. The whole adjuvant enhanced immunization and the last immunization without adjuvant. The last 1 times to strengthen the peripheral venous blood collection of 2mL after 5-7 days of immunization, the specific antibodies in serum were detected by Western blot, and the titer of serum antibody was measured by indirect ELISA method.
Determination of titer by 2.6 ELISA method
The purified fusion protein MCP was 5 mu g/ml, 50 mu l/ hole, and the enzyme labeled plate was coated with different multiple diluent serum. After PBST flushing and HRP labeled Sheep anti rabbit IgG two, the serum was incubated with the TMB substrate solution, and the enzyme labeled instrument was used to determine OD450nm, which was equal to the positive of the negative control hole 2.1 times.
Purification of 2.7 Rabbit anti fusion protein MCP antibody
Primary purified antibody of 2.7.1 saturated ammonium sulfate
Adding equal amount of PBS (pH7.4) to 10ml immunized rabbit serum, adding an equal amount of saturated ammonium sulphate solution to 30min at 4 degrees, centrifuging 15 min at 4 centigrade, dissolving the supernatant, settling the precipitation with 20 ml pre cooled PBS, adding 10mL saturated ammonium sulfate solution, and placing the refrigerators 30min of 4 centigrade, 2000rpm centrifugal 15min, collecting precipitation and precipitating pre cooled dissolution The solution was obtained, and the primary purified IgG antibody solution was obtained.
Purification of antibodies by specific purification of 2.7.2 antigen
First, the CNBr-activated Sepharose 4B chromatography column was pre balanced with the carbonate buffer of PH8.3, the purified MCP protein 5mg was added to 4 C for the night. The unbound protein was removed by the centrifugation on the next day, and the non specific binding site was closed to the bed of crude pure antibody 5mL at 4 degrees centigrade for the night. After the eluate was centrifuged to the column, the absorption of the supernatant was the energy and the recombinant protein MCP. The Rabbit anti MCP antibody was specifically added. Add equal volume glycerin and sodium azide to the final concentration of 0.02%, and then store at -70 C after loading.
Identification of anti MCP antibody in 2.8 rabbits
Western blot detected the antibody specific MCP recombinant protein. Helicobacter pylori, Helicobacter pylori, and the outer membrane protein CSPs of Helicobacter pylori were electrophoretic after SDS-PAG gel electrophoresis and transferred to PVDF membrane. The closed liquid (50 mmol/L Tris-Buffered-Saline, pH7.5, 1%BSA and 0.1% Tween20) oscillates closed 2H at 37 C and then diluted with closed liquid ( 1:500) the Rabbit anti MCP purified antibody was incubated at 4 C for night, after washing the film with 1:5000 diluted HRP- Sheep anti rabbit IgG (Wuhan doctorate company product) at 37 C, incubated for 1h, and after full washing, ECL glowing and developing.
Result
1, isolation and identification of Helicobacter pylori
3 days after the microaerobic culture of the suspicious bacteria on the selective blood plate of Campylobacter jejuni, the size of the needle tip, the mist like transparent colony, the bacterial smear gram blue staining (Figure 3-1), the bacterial gram-negative staining was found to be "S" or "C", similar to Helicobacter pylori, but the bacteria were more elongated and more spiral. Biochemical identification showed bacterial urease experiment Positive, oxidase, peroxidase test positive, nitrate reductase negative. 16S rRNA was amplified by specific primers of Helicobacter and specific primers of Helicobacter pylori, 1.5% agarose gel electrophoresis, 780b positive bands (FIGS. 3-2, 3-3). The results of the product were compared with the homology of genomic DNA in the GeneBank library, and the results were with Helicobacter Bilis. The ATCC43879 homology reached 99%, which confirmed that the bacteria isolated from the experiment were biliary type Helicobacter (Fig. 3-4).
After 3 days of culture of Helicobacter pylori and Helicobacter pylori on the blood plate of Campylobacter jejuni, the size of the needle tip and the foggy transparent colonies were visible.
2, preparation of epicardial protein CSPs of bacterial strain
The bacterial outer membrane protein was prepared by SDS-PAGE electrophoresis after enrichment, and the result of Kaumas brilliant blue staining was shown in Figure 3-5.
3, the induced expression of recombinant fusion protein
After IPTG induction, the recombinant plasmid containing Escherichia coli E.coli BL21 (DE3) could express the recombinant protein rhMCP of relative molecular mass M:14118D. In the previous trial, rhMCP was found to exist in the form of insoluble inclusion bodies. A specific band was found at about 14 KD via Western blot.
4, the separation and purification of recombinant protein
Because the inducible expression of rhMCP is in the form of insoluble inclusion body, the inclusion body is dissolved with 8M urea, and then the gradient urea elution is carried out according to the purification method of denatured protein. The elution effect of UrNTA200 (imidazole concentration is 200mM) is the best. The purified rhMCP is refolding with the gradient urea -PBS dialysis with the dialysis bag, and the refolding is soluble. RhMCP, the protein concentration was measured by BCA method 2.08mg/ml.
5, preparation and purification of Rabbit anti MCP polyclonal antibody
The purified fusion protein His-MCP was used as antigen to immunize the rabbit, the polyclonal antiserum obtained from the rabbit was detected by Western blotting and EL1SA (Figure 3-6, figure 3-7). It was found that there was a specific band in 14KD and the titer of antiserum was 1:32000. It was proved that the high specific and high effective Rabbit anti MCP polyclonal antibody was successfully prepared by the experiment.
6, Western blotting further analyzed the antibody specific bacterial outer membrane protein CSPs and MCP for SDS-PAGE electrophoresis, then transferred to the PVDF membrane, followed by the preparation of the first antibody and the Sheep anti rabbit two resistance, ECL luminescence, film photosensitivity, development, and the specific bands of Helicobacter and MCP protein at 14KD, and Helicobacter pylori and pyloric helicobacter pylori and Helicobacter pylori. The bacterium has no stripe, indicating that the prepared antibody has good activity and can be specifically combined with immunogen (Fig. 3-8).
conclusion
In this study, we successfully isolated the Helicobacter pylori from the colon of Balb/c mice. The purified recombinant protein MCP was used to immunize New Zealand white rabbits, and the anti MCP polyclonal antibody serum was successfully obtained. The antibody was highly specific. After purification, only one specific band was detected with Western blotting, and the potency was 1:32000 (ELISA method). Then, the antibody was then followed by the purified MCP (ELISA method). In the experiment, we found that the antibody was used for the detection of bacterial outer membrane protein CSPs by Western blotting, only the positive band of Helicobacter spp. showed that the antibody had no cross reaction with other common Helicobacter species. Therefore, this experiment provides an important experiment for further study of the epidemiology of Helicobacter pylori and the preparation of a simple detection kit. Basics.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

【参考文献】