發(fā)布時(shí)間：2018-05-31 04:42

  本文選題：膽型螺桿菌 + 分離�。� 參考：《南方醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的和意義 肝螺桿菌(Helicobacter hepaticus, Hh)致病機(jī)制及流行病學(xué)特征尚不完全清楚。本研究通過制備肝螺桿菌甲基基團(tuán)接受趨化信號轉(zhuǎn)導(dǎo)蛋白(methyl-accepting chemotaxis signal transduction protein, MCP)兔抗血清,為進(jìn)一步探討肝螺桿菌在實(shí)驗(yàn)動(dòng)物及人類的感染及及臨床檢測試劑盒的開發(fā)提供實(shí)驗(yàn)基礎(chǔ)。 材料和方法 1、主要材料：細(xì)菌菌株(肝螺桿菌、幽門螺桿菌),空腸彎曲菌瓊脂基礎(chǔ),弗氏完全/不完全佐劑,辣根過氧化物酶標(biāo)記山羊抗兔IgG。Balb/C小鼠,新西蘭大白兔。 2、方法 2.1細(xì)菌分離及培養(yǎng) 2.1.1膽型螺桿菌的分離及鑒定 參照文獻(xiàn)方法分離膽型螺桿菌。SPF (special pathogene free)級Balb/C小鼠禁食48h后,取腸道組織勻漿,涂布于空腸彎曲菌選擇性血瓊脂培養(yǎng)基(含7%凍融脫纖維羊血,選擇性抗生素萬古霉素10mg/L,多粘菌素B 2500單位/L,二性霉素B 10mg/L, TMP 5mg/L),37℃微需氧條件(5%O2.10%CO2及85%N2)下培養(yǎng)3-5天,每天觀察有無菌落生長。挑取可疑菌落傳代培養(yǎng)。 細(xì)菌鑒定：1、形態(tài)鑒定可疑細(xì)菌涂片革蘭染色,油鏡下觀察細(xì)菌形態(tài)。2、生化鑒定可疑細(xì)菌行快速尿素酶、氧化酶、過氧化物酶及硝酸還原酶實(shí)驗(yàn)(檢測試劑盒由環(huán)凱生化公司提供)3、PCR擴(kuò)增細(xì)菌16srRNA基因?qū)⑼该鳌⑨樇獯笮〉木溆妹藓灩沃罞P管中,PBS沖洗三次后,用細(xì)菌基因組DNA提取試劑盒提取細(xì)菌DNA。以可疑菌基因組DNA為模板,采用螺桿菌屬特異性引物行PCR擴(kuò)增。反應(yīng)條件為94℃10min總變性,94℃30s,54℃30s,72℃55s,共35個(gè)循環(huán),72℃10min總延伸。擴(kuò)增產(chǎn)物于1.5%瓊脂糖凝膠中電泳,電泳條件80V,30min。陽性結(jié)果測序分析(測序工作由英俊生物公司完成)。 2.1.2細(xì)菌傳代培養(yǎng) 取肝螺桿菌、膽型螺桿菌、幽門螺桿菌保種液各100μl涂布于空腸彎曲菌選擇性血瓊脂培養(yǎng)基,37℃微需氧條件下培養(yǎng)3天,細(xì)菌涂片革蘭染色觀察細(xì)菌生長情況。 2.2提取細(xì)菌外膜蛋白(cell surface proteins,CSPs) 細(xì)菌在空腸彎曲菌選擇性血瓊脂培養(yǎng)基培養(yǎng)(培養(yǎng)條件同前)48-72h后,用滅菌雙蒸水洗刮下菌苔,室溫4000 r/min,20 min離心洗滌菌液。重復(fù)洗滌一次。棄上清,收集菌泥,稱重,按每4g菌泥加入0.2mol /L、pH 2.2甘氨酸-鹽酸緩沖液100 ml,4℃磁力攪拌30 min,后在4℃條件下12000 r/min,離心15 min。透析,冷凍干燥法濃縮。BCA法測蛋白濃度。 2.3 MCP基因在大腸桿菌中的表達(dá) 將我室保種的重組質(zhì)粒pET22b+/MCP轉(zhuǎn)化大腸桿菌感受態(tài)BL21(DE3),挑單克隆接種于10ml含氨芐青霉素100μg/ml的LB培養(yǎng)基,37℃200rpm培養(yǎng)3個(gè)半小時(shí)至OD600約為0.6-1.0。轉(zhuǎn)接100μl于10ml含氨芐青霉素100μg/ml的LB培養(yǎng)基中,37℃200rpm培養(yǎng)1.5小時(shí)至OD600約為0.6,加入500mmol/L IPTG至終濃度為1mmol/L,37℃180rpm震蕩培養(yǎng)4小時(shí)。將誘導(dǎo)菌液12000rpm 4℃離心5min,取1ml菌液沉淀進(jìn)行SDS-PAGE鑒定。重組蛋白rhMCP包含表達(dá)載體pET22b(+)中的6×His標(biāo)簽,因此用Western blot對rhMCP的表達(dá)再次進(jìn)行鑒定。 2.4 rhMCP蛋白的純化 取1000ml誘導(dǎo)表達(dá)的菌液,12000rpm 4℃離心5min,菌體沉淀加入1/20細(xì)菌生長體積的細(xì)菌裂解液和PMSF,冰上超聲破碎細(xì)菌,10000rpm 4℃離心15min。棄上清,往沉淀中加入1/20細(xì)菌生長體積的UrNTA-0 Buffer和PMSF,將細(xì)胞懸浮,室溫放置30min, 10000rpm 4℃離心15min,取上清。用30ml的UrNTA-0 Buffer平衡3ml NTA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。15ml UrNTA-0 Buffer洗脫未結(jié)合部分,然后分別用15mlUrNTA-20, UrNTA-40, UrNTA-60, UrNTA-100, UrNTA-200, UrNTA-500洗脫,收集洗脫液,用SDS-PAGE確定目標(biāo)蛋白質(zhì)在洗脫液中的分布情況。用梯度PBS-尿素4℃透析(6M-4M-2M-1M),每4小時(shí)換液一次,最后用PBS 4℃透析24小時(shí)。BCA法測蛋白濃度,-70℃保存。 2.5兔抗重組融合蛋白MCP抗血清的制備 取MCP重組蛋白800μl(約800μg)與800μl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MCP 400μl(約400μg)與400μl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MCP加上等體積弗氏不完全佐劑加強(qiáng)免疫,最后一次免疫不加佐劑。最后1次加強(qiáng)免疫后5-7天耳緣靜脈采血2mL,行Western blot檢測血清中是否有特異性抗體,并用間接ELISA法測定血清中抗體的效價(jià)。檢測到特異性抗體后頸動(dòng)脈插管放血,分離血清,置-80℃保存。 2.6 ELISA法測定效價(jià) 純化的融合蛋白MCP按5μg/ml,50μl/孔,包被酶標(biāo)板,分別加人不同倍數(shù)稀釋的血清,PBST沖洗后加HRP標(biāo)記的羊抗兔IgG二抗孵育,最后加TMB底物液顯色,終止后用酶標(biāo)儀測定OD450nm,以等于陰性對照孔的2.1倍者判為陽性。 2.7兔抗融合蛋白MCP抗體的純化 2.7.1飽和硫酸銨法初純抗體 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30min; 4℃2000rpm/min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; 2000rpm離心15min,收集沉淀,沉淀用20ml預(yù)冷的PBS溶解,獲得初步純化的IgG抗體溶液。 2.7.2抗原特異性純化法純化抗體 先用PH8.3的碳酸鹽緩沖液預(yù)平衡CNBr-activated Sepharose 4B層析柱,加入純化的MCP蛋白5mg,4℃過夜。次日離心去除未結(jié)合的蛋白,封閉非特異結(jié)合位點(diǎn)后加入粗純抗體5mL于4℃搖床過夜。向柱中加入洗脫液離心后,吸取上清即為能與重組蛋白MCP特異結(jié)合的兔抗MCP抗體。加入等體積的甘油及疊氮鈉至終濃度0.02%,分裝后-70℃保存。 2.8兔抗MCP抗體的特性鑒定 Western blot檢測抗體特異性取MCP重組蛋白、肝螺桿菌、幽門螺桿菌、及膽型螺桿菌菌體外膜蛋白CSPs變性后行SDS-PAG凝膠電泳,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% Tween20)于37℃振蕩封閉2h,再加入以封閉液稀釋(1：500)的兔抗MCP純化抗體,于4℃孵育過夜,洗膜后加1：5000稀釋的HRP-羊抗兔IgG(武漢博士德公司產(chǎn)品)于37℃溫育1h,充分洗滌后ECL發(fā)光,顯影。 結(jié)果 1、膽型螺桿菌的分離及鑒定 可疑細(xì)菌在空腸彎曲菌選擇性血平板上微需氧培養(yǎng)3天后,肉眼可見針尖大小、霧狀透明菌落,細(xì)菌涂片革蘭染色(圖3-1),見細(xì)菌革蘭染色陰性,呈“S”或“C”形,與幽門螺桿菌相似,但菌體更細(xì)長,螺旋也較多。生化鑒定示細(xì)菌尿素酶實(shí)驗(yàn)陽性,氧化酶、過氧化物酶實(shí)驗(yàn)陽性,硝酸還原酶陰性。采用螺桿菌屬特異性引物及膽型螺桿菌特異性引物擴(kuò)增16s rRNA,1.5%瓊脂糖凝膠電泳,于780b有陽性條帶(圖3-2,圖3-3),產(chǎn)物測序結(jié)果與GeneBank庫中基因組同源性對比,結(jié)果與Helicobacter bilis ATCC43879同源性達(dá)到99%,證實(shí)本實(shí)驗(yàn)所分離的細(xì)菌即為膽型螺桿菌(圖3-4)。 肝螺桿菌、膽型螺桿菌及幽門螺桿菌在空腸彎曲菌血平板上培養(yǎng)3天后,肉眼可見針尖大小、霧狀透明菌落。 2、細(xì)菌菌體外膜蛋白CSPs的制備 制備的細(xì)菌外膜蛋白經(jīng)過濃縮后行SDS-PAGE電泳,考馬斯亮藍(lán)染色結(jié)果如圖3-5所示。 3、重組融合蛋白的誘導(dǎo)表達(dá) 經(jīng)IPTG誘導(dǎo)后,含重組質(zhì)粒的大腸桿菌E.coli BL21(DE3)能夠表達(dá)相對分子質(zhì)量M:14118D的重組蛋白rhMCP,前期試驗(yàn)鑒定rhMCP是以不可溶性的包涵體形式存在。經(jīng)Western blot鑒定,在約14 KD處可見一特異性條帶。 4、重組蛋白的分離純化 由于誘導(dǎo)表達(dá)的rhMCP是以不可溶的包涵體形式存在,因此用8M尿素溶解包涵體,再按變性蛋白純化方法進(jìn)行梯度尿素洗脫,其中以UrNTA200(咪唑濃度為200mM)洗脫效果最佳,純化后的rhMCP用透析袋進(jìn)行梯度尿素-PBS透析復(fù)性,復(fù)性后得到可溶性rhMCP,用BCA法測蛋白濃度為2.08mg/ml。 5、兔抗MCP多克隆抗體的制備純化 用純化的融合蛋白His-MCP作抗原,免疫大白兔,所得的多克隆抗血清,行Western blotting及EL1SA檢測(圖3-6,圖3-7),可見14KD處有特異性條帶,抗血清的效價(jià)為1：32000,證明該實(shí)驗(yàn)制備成功高特異性、高效價(jià)的兔抗MCP多克隆抗體。 6、Western blotting進(jìn)一步分析抗體特異性用制備的細(xì)菌外膜蛋白CSPs及MCP行SDS-PAGE電泳后,轉(zhuǎn)移至PVDF膜上,依次加入制備的一抗及羊抗兔二抗后,ECL發(fā)光,膠片感光、顯影,見肝螺桿菌及MCP蛋白在14KD處可見特異性條帶,而膽型螺桿菌及幽門螺桿菌無條帶,表明所制備的抗體具有較好的活性,可與免疫原特異性結(jié)合(圖3-8)。 結(jié)論 本研究中成功從Balb/c小鼠結(jié)腸中分離出膽型螺桿菌。利用分離純化后的重組蛋白MCP免疫新西蘭大白兔,成功獲得抗MCP的多克隆抗體血清,該抗體特異性強(qiáng),純化后用Western blotting檢測只有一條特異性條帶,且效價(jià)為1：32000(ELISA法)。再后來的實(shí)驗(yàn)中我們發(fā)現(xiàn)該抗體用于Western blotting檢測細(xì)菌外膜蛋白CSPs,只有肝螺桿菌組有陽性條帶,說明該抗體與其他常見螺桿菌屬無交叉反應(yīng)。故本實(shí)驗(yàn)為進(jìn)一步深入研究肝螺桿菌的流行病學(xué)及制備簡便的檢測試劑盒提供了重要的實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R378

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本文編號：1958325