汉坦病毒重组核蛋白的表达及其应用研究

发布时间：2018-01-01 22:13

  本文关键词：汉坦病毒重组核蛋白的表达及其应用研究　出处：《天津医科大学》2008年硕士论文　论文类型：学位论文

  更多相关文章： 汉坦病毒 重组核蛋白 表达 镍亲和层析 ELISA IFA 鼠肺 监测

【摘要】： 【目的】 以汉坦病毒SEO型L99株S基因的重组株E.coli BL21(DE3)/pET32a-L99S为研究对象,进行优化表达、纯化其编码的重组核蛋白(rNP),并制备免疫血清,建立免疫学方法用于检测病人的血清抗体和动物肺组织中的抗原。 【方法】 1.优化E.coli BL21(DE3)/pET32a-L99S的rNP表达条件,观察3个因素(温度、时间、IPTG浓度)18个条件对rNP表达情况的影响,用SDS-PAGE分析目的蛋白的浓度、占菌体总蛋白的百分含量及溶解情况。 2.E.coli BL21(DE3)/pET32a-L99S超声破碎后,包涵体经洗涤和增溶,取其上清部分进行镍亲和层析纯化,收集目的蛋白峰,进一步透析和浓缩后将所获纯化蛋自经SDS-PAGE分析纯度并用Bradford法测定其浓度,用Western-blot鉴定其抗原性。 3.用纯化的rNP免疫日本大耳白兔,制备免疫血清,ELISA测定血清效价,Western-blot鉴定其免疫原性。 4.纯化的rNP用于三种不同的ELISA:rNP-ELISA、HRP-rNP-ELISA和免疫磁性微球ELISA(IMMS-rNP-ELISA),选用HFRS病人阳性血清和健康人血清,比较不同方法的灵敏度和特异度。 5.分别以兔rNP抗血清和HFRS病人混合血清为一抗用于间接免疫荧光法(IFA)检测59份鼠肺组织标本中的HV抗原。将两种IFA检测结果不一致的鼠肺标本采用RT-nested-PCR比较其结果。 【结果】 1.确定rNP表达的最佳条件,即IPTG 0.5 mmol/L、30℃诱导5h,其表达量可达626.04μg/ml,占菌体总蛋白的52.2%,表达产物主要以包涵体形式存在。 2.超声处理后,用镍亲和层析纯化rNP,在连续梯度洗脱中收集咪唑浓度在250-300 mmol/L的洗脱液,所获rNP纯度达95%以上,蛋白浓度为18.01mg/ml,总回收率为28.9%,提纯倍数1.83,Western-blot显示纯化蛋白与HFRS病人血清反应有良好的抗原性。 3.纯化rNP制备抗血清,抗体效价达512 000以上。Western blot显示:纯化的rNP与兔rNP抗血清反应条带单一。 4.三种ELISA检测病人血清IgG抗体,rNP-ELISA,HRP-rNP-ELISA法和IMMS-rNP-ELISA的灵敏度分别为100%、92.5%、100%,特异度分别为95%、100%、100%。 5.用两种IFA检测59份鼠肺冰冻切片的HV抗原,一抗为兔rNP抗血清的IFA有14份阳性,占23.7%;一抗为患者混和血清的IFA有10份阳性,占16.9%。兔rNP抗血清IFA的切片背景清晰,易于判断。将上述两种IFA检测结果不一致的6份鼠肺标本进行RT-nested-PCR,均与兔rNP抗血清IFA的结果相一致。 【结论】 1.确定了重组菌E.coli BL21(DE3)/pET32a-L99S的rNP最佳表达和纯化条件。 2.以纯化的rNP作为抗原,建立了rNP-ELISA、HRP-rNP-ELISA和IMMS-rNP-ELISA检测病人血清HV IgG抗体,灵敏、特异、方法简便、易于推广。 3.用纯化rNP制备兔免疫血清,用于鼠肺组织HV抗原的检测,具有很好的应用前景。
[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.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R373

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