本文關(guān)鍵詞：漢坦病毒重組核蛋白的表達及其應(yīng)用研究　出處：《天津醫(yī)科大學》2008年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 漢坦病毒 重組核蛋白 表達 鎳親和層析 ELISA IFA 鼠肺 監(jiān)測

【摘要】： 【目的】 以漢坦病毒SEO型L99株S基因的重組株E.coli BL21(DE3)/pET32a-L99S為研究對象,進行優(yōu)化表達、純化其編碼的重組核蛋白(rNP),并制備免疫血清,建立免疫學方法用于檢測病人的血清抗體和動物肺組織中的抗原。 【方法】 1.優(yōu)化E.coli BL21(DE3)/pET32a-L99S的rNP表達條件,觀察3個因素(溫度、時間、IPTG濃度)18個條件對rNP表達情況的影響,用SDS-PAGE分析目的蛋白的濃度、占菌體總蛋白的百分含量及溶解情況。 2.E.coli BL21(DE3)/pET32a-L99S超聲破碎后,包涵體經(jīng)洗滌和增溶,取其上清部分進行鎳親和層析純化,收集目的蛋白峰,進一步透析和濃縮后將所獲純化蛋自經(jīng)SDS-PAGE分析純度并用Bradford法測定其濃度,用Western-blot鑒定其抗原性。 3.用純化的rNP免疫日本大耳白兔,制備免疫血清,ELISA測定血清效價,Western-blot鑒定其免疫原性。 4.純化的rNP用于三種不同的ELISA:rNP-ELISA、HRP-rNP-ELISA和免疫磁性微球ELISA(IMMS-rNP-ELISA),選用HFRS病人陽性血清和健康人血清,比較不同方法的靈敏度和特異度。 5.分別以兔rNP抗血清和HFRS病人混合血清為一抗用于間接免疫熒光法(IFA)檢測59份鼠肺組織標本中的HV抗原。將兩種IFA檢測結(jié)果不一致的鼠肺標本采用RT-nested-PCR比較其結(jié)果。 【結(jié)果】 1.確定rNP表達的最佳條件,即IPTG 0.5 mmol/L、30℃誘導5h,其表達量可達626.04μg/ml,占菌體總蛋白的52.2%,表達產(chǎn)物主要以包涵體形式存在。 2.超聲處理后,用鎳親和層析純化rNP,在連續(xù)梯度洗脫中收集咪唑濃度在250-300 mmol/L的洗脫液,所獲rNP純度達95%以上,蛋白濃度為18.01mg/ml,總回收率為28.9%,提純倍數(shù)1.83,Western-blot顯示純化蛋白與HFRS病人血清反應(yīng)有良好的抗原性。 3.純化rNP制備抗血清,抗體效價達512 000以上。Western blot顯示:純化的rNP與兔rNP抗血清反應(yīng)條帶單一。 4.三種ELISA檢測病人血清IgG抗體,rNP-ELISA,HRP-rNP-ELISA法和IMMS-rNP-ELISA的靈敏度分別為100%、92.5%、100%,特異度分別為95%、100%、100%。 5.用兩種IFA檢測59份鼠肺冰凍切片的HV抗原,一抗為兔rNP抗血清的IFA有14份陽性,占23.7%;一抗為患者混和血清的IFA有10份陽性,占16.9%。兔rNP抗血清IFA的切片背景清晰,易于判斷。將上述兩種IFA檢測結(jié)果不一致的6份鼠肺標本進行RT-nested-PCR,均與兔rNP抗血清IFA的結(jié)果相一致。 【結(jié)論】 1.確定了重組菌E.coli BL21(DE3)/pET32a-L99S的rNP最佳表達和純化條件。 2.以純化的rNP作為抗原,建立了rNP-ELISA、HRP-rNP-ELISA和IMMS-rNP-ELISA檢測病人血清HV IgG抗體,靈敏、特異、方法簡便、易于推廣。 3.用純化rNP制備兔免疫血清,用于鼠肺組織HV抗原的檢測,具有很好的應(yīng)用前景。
[Abstract]:[Objective]
The SEO of hantavirus L99 strain S gene recombinant strain E.coli BL21 (DE3) /pET32a-L99S as the research object, to optimize the expression of recombinant nucleocapsid protein purification encoding (rNP), and preparation of immune serum, establishment of immunological methods for the detection of the patient's serum antibody and animal lung tissue antigens.
[method]
1. optimization of E.coli BL21 (DE3) expression condition of /pET32a-L99S rNP, to observe the 3 factors (temperature, time, concentration of IPTG) the impact of the 18 conditions on the expression of rNP protein by SDS-PAGE analysis of concentration, percentage of content and dissolution of the total bacterial protein.
2.E.coli BL21 (DE3) /pET32a-L99S after ultrasonication, the inclusion bodies were washed and solubilized, take the supernatant of nickel affinity chromatography, the purpose of collecting protein peaks, further dialysis and concentration will be obtained after the purification of eggs from SDS-PAGE purity analysis and determination of the concentration of Bradford, Western-blot was used to identify its antigenicity.
3. immunized Japanese big ear rabbits with purified rNP, immunized serum was prepared, serum titer was measured by ELISA, and Western-blot was identified as immunogenicity.
4. purified rNP was applied to three different ELISA:rNP-ELISA, HRP-rNP-ELISA and immunomagnetic microspheres ELISA (IMMS-rNP-ELISA). HFRS positive serum and healthy human serum were selected to compare the sensitivity and specificity of different methods.
5., using rabbit rNP antiserum and mixed serum of HFRS patients as one antibody, indirect immunofluorescence assay (IFA) was used to detect HV antigen in 59 lung tissues of rats. The results of two lung IFA samples with inconsistent results were compared by RT-nested-PCR.
[results]
1. determine the optimum conditions for the expression of rNP, IPTG 0.5 mmol/L, 30 C 5h induced the expression of up to 626.04 g/ml, the total bacterial protein 52.2% expression products mainly existed in the form of inclusion body.
2. after ultrasonic treatment, rNP was purified by nickel affinity, collect the imidazole concentration in the eluent 250-300 mmol/L in continuous gradient elution, the purity of rNP was above 95%, the protein concentration was 18.01mg/ml, the total recovery rate was 28.9%, purification factor 1.83, Western-blot showed the purified serum protein and HFRS patients have good antigenicity.
3. purified rNP was prepared for antiserum, and the antibody titer was over 512000.Western blot. The purified rNP and rabbit rNP antiserum were single.
4., three kinds of ELISA were used to detect the serum IgG antibody. The sensitivity of rNP-ELISA, HRP-rNP-ELISA and IMMS-rNP-ELISA were 100%, 92.5%, 100%, and the specificity was 95%, 100%, 100%. respectively.
5. HV two IFA 59 antigen detection of rat lung frozen sections, an anti rabbit antiserum against rNP IFA 14 were positive, accounting for 23.7%; a mixture of anti patients serum IFA 10 were positive, accounting for 16.9%. of rabbit antiserum against IFA rNP slice background clear, easy to judge the above two IFA. The detection results of 6 rat lung specimens were not consistent with RT-nested-PCR, consistent results with rabbit rNP antiserum of IFA.
[Conclusion]
1. the optimum expression and purification conditions for the rNP of recombinant strain E.coli BL21 (DE3) /pET32a-L99S were determined.
2., using purified rNP as antigen, we established rNP-ELISA, HRP-rNP-ELISA and IMMS-rNP-ELISA to detect serum IgG antibody in patients. It is sensitive, specific, simple and easy to promote.
3. the immunized serum of rabbit is prepared by purified rNP, which is used for the detection of HV antigen of rat lung tissue, which has a good prospect of application.

【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R373

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