本文選題：單鏈抗體　切入點：煤工塵肺　出處：《鄭州大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：背景和目的 煤工塵肺是塵肺病的一種,可引起患者長期慢性并且不可逆的肺組織纖維化。近年來雖然對塵肺病的發(fā)生發(fā)展過程研究很多,但是其機制還不十分清楚,尚未找到消除和逆轉(zhuǎn)其所致纖維化的方法。大量研究表明塵肺病的發(fā)生發(fā)展過程與機體免疫功能的變化密切相關(guān),但以往由于技術(shù)限制,沒有對塵肺病免疫抗體進行系統(tǒng)的觀察研究。隨著分子免疫、細胞免疫技術(shù)的快速發(fā)展,噬菌體抗體庫技術(shù)得到了極大的提高,并廣泛應(yīng)用于生命科學(xué)的許多領(lǐng)域,尤其是在腫瘤和自身免疫性疾病的應(yīng)用中取得了滿意的效果,但在塵肺病的研究尚未報道。該課題擬用課題組已構(gòu)建的人源性煤工塵肺噬菌體單鏈抗體庫,從中篩選煤工塵肺特異性單鏈抗體(single chain variable fragment, scFv),并采用體外實驗和人群流行病學(xué)調(diào)查進行初步驗證,尋找體內(nèi)是否存在煤工塵肺抗體生物標志,為塵肺病免疫學(xué)機制研究提供依據(jù)。 方法 1.煤工塵肺噬菌體單鏈抗體庫的富集篩選：利用煤工塵肺患者和對照者肺灌洗液,對已構(gòu)建好的人源性煤工塵肺噬菌體單鏈抗體庫進行4輪富集篩選,并測定庫容量。每輪富集篩選時隨機挑取20個單菌落,分別制備成scFv。 2.煤工塵肺噬菌體scFv特異性的檢測：將制備好的scFv包被到酶標板上,Elisa檢測煤工塵肺患者(P)和對照者(N)肺灌洗液OD450值,以P/N2.0為陽性克隆,P/N3.0為強陽性克隆。 3.強陽性克隆質(zhì)粒scFv基因的驗證：提取強陽性克隆質(zhì)粒DNA,進行PCR擴增和雙酶切驗證是否插入scFv基因。 4.強陽性克隆的鑒定：強陽性克隆質(zhì)粒PCR擴增后進行測序,翻譯為蛋白序列,然后進行比對,鑒定為何種scFv。 5.可溶性抗體的誘導(dǎo)表達：強陽性克隆感染大腸桿菌HB2151,經(jīng)IPTG誘導(dǎo)表達可溶性抗體,并進行SDS-PAGE進行驗證。 6.建立Gz-B抗體抑制的大鼠肺纖維化體外細胞模型。空白組為無游離二氧化硅刺激的巨噬細胞上清刺激大鼠肺成纖維細胞,游離二氧化硅處理組為游離二氧化硅刺激的巨噬細胞上清刺激大鼠肺成纖維細胞,抑制組為游離二氧化硅刺激的巨噬細胞上清刺激Gz-B抗體抑制的大鼠肺成纖維細胞。建立Gz-B抗體抑制的大鼠肺纖維化體外細胞模型后,分別用實時定量PCR和Elisa方法檢測空白組、游離二氧化硅處理組和抑制組a-SMA、 Col Ⅰ和Col Ⅲ mRNA和蛋白的表達水平。 7.選擇159例男性在崗接塵工人作為接塵組和85例男性崗前體檢者作為對照組,Elisa法檢測血漿中篩選出的強陽性克隆抗體濃度。 結(jié)果 1.煤工塵肺噬菌體單鏈抗體庫的富集篩選：隨著富集篩選次數(shù)的增加,煤工塵肺噬菌體抗體庫的產(chǎn)出量逐漸增多,從8.9×103pfu增加到5.5x106pfu,并且投入產(chǎn)出比從1.46×10-6上升到6.40×10-4,富集倍數(shù)增加了438.36倍,說明煤工塵肺噬菌體抗體得到了有效的富集。 2.煤工塵肺噬菌體scFv特異性的檢測：隨著富集篩選次數(shù)的增加,P/N值和陽性克隆率都隨之增加,說明了煤工塵肺噬菌體抗體的特異性逐漸增強。 3.強陽性克隆質(zhì)粒scFv基因的驗證：共有6個煤工塵肺噬菌體scFv為強陽性克隆,PCR結(jié)果可見擴增片段大小約為900bp,雙酶切結(jié)果可見750bp的scFv基因片段和剩余的載體片段,均證明了強陽性克隆插入了scFv基因。 4.比對鑒定6個強陽性克隆分別為血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)、白介素-18(interleukin-18, IL-18)、熱休克蛋白-70.1(heat shock protein-70.1, HSP70.1)、人表皮生長因子受體3(human epidermal growth factor receptor3, HER3)、顆粒酶B(Granzyme B, Gz-B)和類風濕因子(rheumatoid factor. RF) scFv。 5.強陽性克隆SDS-PAGE結(jié)果可見32kD處有條帶出現(xiàn),說明scFv實現(xiàn)了可溶性表達。 6.空白組、游離二氧化硅處理組和抑制組α-SMA、 Coll和Col Ⅲ mRNA和蛋白表達水平的差異有統(tǒng)計學(xué)意義(P0.05)。抑制組α-SMA、 Col Ⅰ和Col ⅢmRNA和蛋白的表達水平均明顯高于空白組和游離二氧化硅處理組,差異也有統(tǒng)計學(xué)意義(P0.05)。 7.接塵5年組人群血漿VEGF、 HSP70.1、HER3和RF抗體濃度高于對照組,而VEGF、 HER3和Gz-B抗體陽性率高于對照組,差異均有統(tǒng)計學(xué)意義(P0.017)；接塵≥5年組人群6種抗體濃度和陽性率均明顯高于對照組,且差異有統(tǒng)計學(xué)意義(P0.017)；接塵≥5年組人群血漿6種抗體濃度均明顯高于5年組,且差異有統(tǒng)計學(xué)意義(P0.017)。 結(jié)論 1.從已構(gòu)建的人源性煤工塵肺噬菌體抗體庫篩選出6種煤工塵肺相關(guān)抗體,并且實現(xiàn)了可溶性表達。 2.建立Gz-B抗體抑制的大鼠肺纖維化體外細胞模型,并發(fā)現(xiàn)Gz-B高表達可能與塵肺發(fā)生有關(guān)。 3.粉塵接觸可以導(dǎo)致接塵工人6種抗體濃度和陽性率升高,且隨接塵時間的增加而升高。
[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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R135.2

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