煤工尘肺噬菌体单链抗体的筛选和初步验证

发布时间：2018-03-20 19:12

本文选题：单链抗体　切入点：煤工尘肺　出处：《郑州大学》2012年硕士论文　论文类型：学位论文

【摘要】：背景和目的煤工尘肺是尘肺病的一种,可引起患者长期慢性并且不可逆的肺组织纤维化。近年来虽然对尘肺病的发生发展过程研究很多,但是其机制还不十分清楚,尚未找到消除和逆转其所致纤维化的方法。大量研究表明尘肺病的发生发展过程与机体免疫功能的变化密切相关,但以往由于技术限制,没有对尘肺病免疫抗体进行系统的观察研究。随着分子免疫、细胞免疫技术的快速发展,噬菌体抗体库技术得到了极大的提高,并广泛应用于生命科学的许多领域,尤其是在肿瘤和自身免疫性疾病的应用中取得了满意的效果,但在尘肺病的研究尚未报道。该课题拟用课题组已构建的人源性煤工尘肺噬菌体单链抗体库,从中筛选煤工尘肺特异性单链抗体(single chain variable fragment, scFv),并采用体外实验和人群流行病学调查进行初步验证,寻找体内是否存在煤工尘肺抗体生物标志,为尘肺病免疫学机制研究提供依据。方法 1.煤工尘肺噬菌体单链抗体库的富集筛选：利用煤工尘肺患者和对照者肺灌洗液,对已构建好的人源性煤工尘肺噬菌体单链抗体库进行4轮富集筛选,并测定库容量。每轮富集筛选时随机挑取20个单菌落,分别制备成scFv。 2.煤工尘肺噬菌体scFv特异性的检测：将制备好的scFv包被到酶标板上,Elisa检测煤工尘肺患者(P)和对照者(N)肺灌洗液OD450值,以P/N2.0为阳性克隆,P/N3.0为强阳性克隆。 3.强阳性克隆质粒scFv基因的验证：提取强阳性克隆质粒DNA,进行PCR扩增和双酶切验证是否插入scFv基因。 4.强阳性克隆的鉴定：强阳性克隆质粒PCR扩增后进行测序,翻译为蛋白序列,然后进行比对,鉴定为何种scFv。 5.可溶性抗体的诱导表达：强阳性克隆感染大肠杆菌HB2151,经IPTG诱导表达可溶性抗体,并进行SDS-PAGE进行验证。 6.建立Gz-B抗体抑制的大鼠肺纤维化体外细胞模型。空白组为无游离二氧化硅刺激的巨噬细胞上清刺激大鼠肺成纤维细胞,游离二氧化硅处理组为游离二氧化硅刺激的巨噬细胞上清刺激大鼠肺成纤维细胞,抑制组为游离二氧化硅刺激的巨噬细胞上清刺激Gz-B抗体抑制的大鼠肺成纤维细胞。建立Gz-B抗体抑制的大鼠肺纤维化体外细胞模型后,分别用实时定量PCR和Elisa方法检测空白组、游离二氧化硅处理组和抑制组a-SMA、 Col Ⅰ和Col Ⅲ mRNA和蛋白的表达水平。 7.选择159例男性在岗接尘工人作为接尘组和85例男性岗前体检者作为对照组,Elisa法检测血浆中筛选出的强阳性克隆抗体浓度。结果 1.煤工尘肺噬菌体单链抗体库的富集筛选：随着富集筛选次数的增加,煤工尘肺噬菌体抗体库的产出量逐渐增多,从8.9×103pfu增加到5.5x106pfu,并且投入产出比从1.46×10-6上升到6.40×10-4,富集倍数增加了438.36倍,说明煤工尘肺噬菌体抗体得到了有效的富集。 2.煤工尘肺噬菌体scFv特异性的检测：随着富集筛选次数的增加,P/N值和阳性克隆率都随之增加,说明了煤工尘肺噬菌体抗体的特异性逐渐增强。 3.强阳性克隆质粒scFv基因的验证：共有6个煤工尘肺噬菌体scFv为强阳性克隆,PCR结果可见扩增片段大小约为900bp,双酶切结果可见750bp的scFv基因片段和剩余的载体片段,均证明了强阳性克隆插入了scFv基因。 4.比对鉴定6个强阳性克隆分别为血管内皮生长因子(vascular endothelial growth factor, VEGF)、白介素-18(interleukin-18, IL-18)、热休克蛋白-70.1(heat shock protein-70.1, HSP70.1)、人表皮生长因子受体3(human epidermal growth factor receptor3, HER3)、颗粒酶B(Granzyme B, Gz-B)和类风湿因子(rheumatoid factor. RF) scFv。 5.强阳性克隆SDS-PAGE结果可见32kD处有条带出现,说明scFv实现了可溶性表达。 6.空白组、游离二氧化硅处理组和抑制组α-SMA、 Coll和Col Ⅲ mRNA和蛋白表达水平的差异有统计学意义(P0.05)。抑制组α-SMA、 Col Ⅰ和Col ⅢmRNA和蛋白的表达水平均明显高于空白组和游离二氧化硅处理组,差异也有统计学意义(P0.05)。 7.接尘5年组人群血浆VEGF、 HSP70.1、HER3和RF抗体浓度高于对照组,而VEGF、 HER3和Gz-B抗体阳性率高于对照组,差异均有统计学意义(P0.017)；接尘≥5年组人群6种抗体浓度和阳性率均明显高于对照组,且差异有统计学意义(P0.017)；接尘≥5年组人群血浆6种抗体浓度均明显高于5年组,且差异有统计学意义(P0.017)。结论 1.从已构建的人源性煤工尘肺噬菌体抗体库筛选出6种煤工尘肺相关抗体,并且实现了可溶性表达。 2.建立Gz-B抗体抑制的大鼠肺纤维化体外细胞模型,并发现Gz-B高表达可能与尘肺发生有关。 3.粉尘接触可以导致接尘工人6种抗体浓度和阳性率升高,且随接尘时间的增加而升高。
[Abstract]:Background and purpose
CWP is a kind of pneumoconiosis disease, patients can cause chronic and irreversible pulmonary fibrosis. In recent years, although the incidence of pneumoconiosis during the development of a lot of research, but its mechanism is not very clear, and the method has not been found to eliminate the reversal of fibrosis. Due to a large number of studies show that the occurrence and development of pneumoconiosis and the immune function changes are closely related, but in the past due to technical limitations, no systematic observation on pneumoconiosis antibody. With the rapid development of molecular immunology, cell immune technology, phage display technology has been greatly improved, and is widely used in many fields of life science, especially with satisfactory the effect of application in cancer and autoimmune diseases, but no report on study of pneumoconiosis. The study of human macrophages has been constructed with pneumoconiosis research group Cell single chain antibody library screening CWP scFv from (single chain variable fragment, scFv), and the epidemiological investigation of in vitro experiment and preliminary validation for in vivo populations, the existence of CWP antibody biomarkers, provide the basis for the study on immunological mechanism of pneumoconiosis.
Method
1. pneumoconiosis phage antibody library enrichment screening by the pneumoconiosis patients and controls the bronchoalveolar lavage fluid, the constructed human pneumoconiosis phage antibody library for 4 rounds of panning, and determination of the capacity of the reservoir. Each round of enrichment screening randomly selected 20 single colonies, respectively prepared by scFv.
2., the specificity of phage scFv in coal worker's pneumoconiosis was detected: the prepared scFv package was applied to the enzyme labelling board, Elisa was used to detect OD450 value in lung lavage fluid of coal workers pneumoconiosis patients (P) and controls (N), P/N2.0 was positive clones, P/N3.0 was a strong positive clone.
3. strong positive cloned plasmid scFv gene was verified: the strong positive cloned plasmid DNA was extracted, and PCR amplification and double enzyme digestion were used to verify whether the scFv gene was inserted.
The identification of 4. strong positive clones: strong positive cloned plasmid PCR was amplified and sequenced, translated into protein sequence and then compared to identify scFv.
5. inducible expression of soluble antibody: strong positive clones were infected with Escherichia coli HB2151, induced by IPTG to express soluble antibody, and were verified by SDS-PAGE.
6. to establish a rat model of pulmonary fibrosis in vitro. The blank group Gz-B antibody inhibited the stimulation of rat lung fibroblasts as no free silica stimulated macrophages supernatant, free silica treated group for free silica stimulated macrophages supernatant stimulated rat lung fibroblast cells, inhibiting group free silica stimulated macrophages supernatant stimulated Gz-B antibody inhibition of rat lung fibroblasts. Establishment of rat pulmonary fibrosis model in vitro Gz-B antibody suppression, respectively using real-time quantitative Elisa method for detection of PCR and control group, two oxygen free silicon treatment group and inhibition group a-SMA, the expression level of Col I and Col III and mRNA protein.
7., 159 male dust exposed workers and 85 male pre physical checkers were selected as control group. Elisa was used to detect the concentration of strong positive clones selected from plasma.
Result
1. pneumoconiosis phage antibody library enrichment and screening: with the increase in the number of output enrichment screening, pneumoconiosis phage antibody library gradually increased from 8.9 x 103pfu to 5.5x106pfu, and the input-output ratio increased from 1.46 x 10-6 to 6.40 x 10-4, enrichment ratio increased by 438.36 times, that of coal workers' pneumoconiosis phage antibody has been effectively enriched.
2., the specificity of phage scFv in coal worker's pneumoconiosis was detected: the number of P/N and positive clones increased with the increase of enrichment and screening times, indicating that the specificity of phage antibody of coal pneumoconiosis increased.
Validation of 3. strong positive clones of scFv plasmid: a total of 6 coal workers pneumoconiosis phage scFv strong positive clones, PCR results showed the amplified fragment size is about 900bp, double enzyme digestion results scFv gene fragment visible 750bp and residual vector segment, showed strong positive clones of scFv gene.
4. identify 6 strong positive clones were vascular endothelial growth factor (vascular endothelial, growth factor, VEGF), interleukin -18 (interleukin-18, IL-18), heat shock protein -70.1 (heat shock protein-70.1, HSP70.1), human epidermal growth factor receptor 3 (human epidermal growth factor receptor3, HER3, Granzyme B () Granzyme B, Gz-B) and rheumatoid factor (rheumatoid factor. RF) scFv.
The results of 5. strong positive clones SDS-PAGE showed that there was a strip in 32kD, indicating that scFv realized soluble expression.
6. blank group, the free silica treated group and inhibition group alpha -SMA, there was significant difference in the expression level of Coll and Col in mRNA and protein (P0.05) inhibitory group. Alpha -SMA, expression of Col I and Col III and mRNA protein were significantly higher than the blank group and free silica treated group, there was also significant difference (P0.05).
7. dust 5 years groups of plasma VEGF, HSP70.1, HER3 and RF antibodies were higher than the control group, while VEGF, HER3 and Gz-B antibody positive rate was higher than the control group, the differences were statistically significant (P0.017); the dust is more than 5 years groups of 6 kinds of antibody concentration and positive rate were significantly higher than the control group, there was statistical and the difference (P0.017); the dust concentration is more than 5 years group, 6 groups of plasma antibodies were significantly higher than that in 5 years group, and the difference was statistically significant (P0.017).
conclusion
1. 6 kinds of coal workers' pneumoconiosis related antibodies were screened from the established human pneumoconiosis phage antibody library, and the soluble expression was realized.
2. to establish an in vitro cell model of rat pulmonary fibrosis inhibited by Gz-B antibody, and found that the high expression of Gz-B may be related to the occurrence of pneumoconiosis.
3. dust exposure can increase the concentration and positive rate of 6 kinds of antibody in the workers, and increase with the increasing of the dust time.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R135.2

【参考文献】