Screening And Preliminary Verification Of Phage Single-chain Antibody Library Against Coal Workers’ Pneumoconiosis(National Natural Science Foundation Of China)

Background and objectiveCoal workers’pneumoconiosis (CWP) can cause chronic and irreversible lung fibrosis as a kind of pneumoconiosis. Many scholars carried a variety of studies to understand the occurrence and development of pneumoconiosis, but the mechanism is remains unclear. There is no radical remedy to eliminate or reversal it up to now. Many studies showed that the occurrence and development of pneumoconiosis was closly related to immune function, but there was no systematic observation on pneumoconiosis antibody due to technical limitation in the past. Phage-displayed antibody library technology was widely used in many areas of life sciences with the rapid development of molecular immunology and cellular immunology in recent years, especially in cancers and antoimmune diseases. There is no report on study of pneumoconiosis now. We decided to screen related single chain variable fragment (scFv) against CWP in a human phage antibody library, and preliminary verify by population epidemiological surveys and vitro experiments. It is very significant to search for some immunologic biomarkers against CWP, which can provide basis for immunological mechanism of pneumoconiosis.Methods1. Enrichment and screening of phage-displayed antibody library against CWP. Phage-displayed antibody library against CWP was enriched and screened with lung lavage of CWP patients and controls for four rounds, and library capacity was detected.20single colonies were randomly picked and prepared scFvs at every round.2. Specificity detection of scFvs. ScFvs were coated Elisa board and detected OD450of CWP (P) and control (N) respectively. Positive clone was P/N>2, and strong positive clone was P/N>3.3. Gene verification of strong positive clone. Plasmid DNAs were extracted from strong postive clones, followed by PCR amplification and enzyme digestion to verify insertion of scFv gene.4. Identification of strong positive clone. PCR products of strong positive clones were sequenced and translated into protein sequence. Strong positive clones were identitied with protein comparison.5. Inducible expression of soluble antibody. Strong positive clones were infected with E.coli HB2151and induced with IPTG. Soluble antibodies were verified by SDS-PAGE..6. Establishing rat pulmonary fibrosis models in vitro for Gz-B antibody inhibited. Blank group:AM supernatant without free SiO2stimulated rat fibroblasts. Free SiO2group:AM supernatant with free SiO2stimulated rat fibroblasts. Inhibiting group:AM supernatant with free SiO2stimulated rat fibroblasts treated by Gz-B antibody inhibitor. mRNA and protein levels of a-SMA, Col I and Col III in blank group, free SiO2group and inhibiting group were detected by real-time PCR and Elisa respectively.7. One hundred and fifty nine dust exposed male workers were selected as dust group, and eighty five pre-post male persons were selected as control group. The plasma antibody concentration of strong positive clones in two groups were detected by Elisa.Results1. Enrichment and screening of phage-displayed antibody library against CWP. The output of phage-displayed antibody library gradually increased with screening rounds, from8.9×103pfu to5.5×106pfu. The input-output radio increasd from1.46×10-6to6.40×10-4, and the enrichment factor increased438.36times. It’s showed that phage-displayed antibody library against CWP was got effective enrichment.2. Specificity detection of scFvs. P/N and positive clones rates were increased with screening rounds. It’s showed that specificity of scFvs increased gradually.3. Gene verification of strong positive clone. A total of six scFvs was strong positive clones. The length was about750bp after PCR amplifying, and the enzyme digestion result appeared two bands (scFv gene fragments and remaining vector fragments). It’s showed that scFv gene inserted into strong positive clones.4. Identification of strong positive clone. Six strong positive clones were vascular endothelial growth factor (VEGF), interleukin-18(IL-18), heat shock protein70.1(HSP70.1), human epidermal growth factor receptor3(HER3), Granzyme B (Gz-B) and rheumatoid factor (RF) scFv after protein comparison.5. Inducible expression of soluble antibody. Strong positive clones appeared32kD after SDS-PAGE. It’s showed that scFv achieved inducible expression.6. There were statistically significant differences in mRNA and protein levels of α-SMA, Col Ⅰ and Col Ⅲ in three groups (P<0.05). Compared with blank goup and free SiO2group, mRNA and protein levels of a-SMA, Col Ⅰ and Col Ⅲ increased significantly in inhibiting group (P<0.05).7. Compared with control group, the plasma antibody concentration of VEGF, HSP70.1, HER3and RF increased significantly in<5dust years group (P<0.017), and positive rate of VEGF, HER3and Gz-B ncreased significantly in<5dust years group (P<0.017). Six antibodies concentration and positive rate of≥5dust years group were significantly higher than those of<5dust years group.Compared with<5dust years group, six plasma antibodies concentration increased significantly in≥5dust years group (P<0.05).Conclusion1. Related scFvs against CWP were screened from human phage antibody library and achieved inducible expression.2. Establishing rat pulmonary fibrosis models in vitro for Gz-B antibody inhibited, and increaing expression of Gz-B was related to occurrence of pneumoconiosis.3. Dust exposure could lead to massive release of antibody of IL-18, VEGF, HSP70.1, HER3, Gz-B and RF in dust exposed workers, and the antibody concentration of six antibodies increased with increaing dust exposure time.