Analysis of Epidermal Growth Factor Receptor Gene Mutation in 274 Cases of Non-Small Cell Lung Cancer / 中国药房

OBJECTIVE:To study the gene mutation status of epidermal growth factor receptor(EGFR)in non-small cell lung cancer(NSCLC)patients and its relationship with clinical indexes,and to provide reference for individualized administration of EGFR-TKI in NSCLC patients. METHODS:Totally of 274 NSCLC patients from the northern of Jiangsu area were selected from our hospital during Jan. 2015-Dec. 2017. Mutation status of EGFR gene in lung tissue was determined by amplification refractory mutation system (ARMS)-TaqMan PCR assay. The relationship of EGFR gene mutation with clinical indexes as gender,age, smoking status, staging, tumor differentiation and pathological type were analyzed retrospectively. Compared with related literatures,the regional differences of EGFR gene mutation were analyzed. RESULTS:Among 274 NSCLC patients,112 patients suffered from EGFR gene mutation with total mutation rate of 40.88%. There were 50,57,3,2 cases of exon 19,exon 21,exon 20 and exon 19+21 mutation,and the types of EGFR gene mutation were delE746-A750,L858R and insH773-V774H,etc. The mutation rates of EGFR gene exon 19,exon 21 in non-smoking,early,well-differentiated and adenocarcinoma patients were 52.50%,47.24%,46.36% and 45.00%,which were significantly higher than smoking (28.57%),advanced (27.03%), poor-differentiated(31.71%)and squamous cell carcinoma(27.66%)patients,with statistical significance(P＜0.05). There was no statistical significance in mutation rates of EGFR gene exon 19 and exon 21 between male and female,≥65 year-old and ＜65 year-old patients (P＞0.05). EGFR mutation rate of NSCLC subjects from the northern of Jiangsu area was significantly higher than Shanghai area(P＜0.05);there was no statistical significance compared with Yunnan area(P＞0.05)but mutation types were different. CONCLUSIONS:There is the highest EGFR gene mutation rate in its exon 21,lesser in exon 19,rare in exon 20 and exon 19+21 among NSCLC patients from the Northern of Jiangsu area. There are obvious regional differences. The mutation rate of EGFR gene mutation exon 19 and exon 21 are associated with smoking status,staging,tumor differentiation and pathological type of NSCLC patients. The non-smoking, early stage, well-differentiated and adenocarcinoma patients are more likely to benefit from EGFR-TKI targeted therapy.

Construction and expression of humanized anti-HBsAg scFv targeting interferon-alpha in escherichia coli / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To develop a bacteria expression system to produce the fusion protein of humanized anti-HBsAg scFV and interferon-alpha.</p><p><b>METHODS</b>The expression vector was constructed after cleaving the plasmids harboring the humanized anti-HBsAg scFv and interferon alpha respectively and ligating to linearized pET22b subsequence. The expression of fusion protein in E.coli was analyzed by SDS-PAGE. The binding activity and antiviral activity of the fusion protein was characterized by competing inhibition test and cytopathic effect reduction.</p><p><b>RESULTS</b>The plasmid harboring the in frame arranged fusion gene was constructed and identified. After induction for 12h, a new band close to 4.5 10(4) was observed using SDS-PAGE. Results of competing ELISA and cytopathic effect reduction showed the fusion protein retained its specific binding activity and antiviral activities.</p><p><b>CONCLUSIONS</b>The construction and expression of the fusion gene of humanized anti-HBsAg scFv and interferon in E.coli are successful.</p>

Screening and application of human-derived HBsAg bacteriophage single chain antibody in clinical diagnosis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To identify human single chain Fv antibody (ScFv) against hepatitis B viral surface antigen.</p><p><b>METHODS</b>The recombinant phages were panned by HBsAg which was coated in a microtiter plate, after five rounds of biopanning, 56 phage clones were identified specific to HBsAg. The specificity of ScFv was evaluated by ELISA and immunohistochemistry, respectively.</p><p><b>RESULTS</b>The data of HB sAg-ScFv DNA digestion and DNA sequencing showed that the ScFv gene is composed of 750 bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitis B surface antigen has a specific combination character with hepatitis B surface antigen of different sources and paraffin-embedded patients tissue specimens, it did not react with normal liver tissue and HCV.</p><p><b>CONCLUSIONS</b>The application of HBsAg specific ScFv in immunohistochemistry was successfully achieved.</p>

Quasispecies and variations of hepatitis B virus: core promotor region as an example / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the HBV quasispecies groups in the patients with chronic HBV infection.</p><p><b>METHODS</b>A set of specific primers was synthesized according to DNA sequence of HBV strain found in China. The whole core promoter (CP) region was amplified by PCR method from the sera of 3 patients with chronic HBV infection, and the PCR products were subcloned into pGEM Teasy vectors. The clones were randomly selected to be sequenced. Sequence comparison of the selected clones was made to find the difference.</p><p><b>RESULTS</b>By comparison, it was found that each sequence of selected clones was different. The point mutation always occurred in TATA-like boxes, especially from T to C replacement on 184 site. There is a hot region (33.3% 5/15) in basic core promoter where deletion mutation frequently happened.</p><p><b>CONCLUSIONS</b>There is a hot deletion region near DR I in CP. The replacement at 184 nt (T to C) in the third TATA-like box may influence the expression of pre-C/C protein. The sequencing results suggest that there are HBV quasispecies groups in chronically infected patients.</p>

Screening and cloning gene of hepatocyte protein interacting with hepatitis C virus core protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To clone the unknown gene of hepatocyte protein interacting with hepatitis C virus core protein.</p><p><b>METHODS</b>Using the yeast dual hybrid system 3, bait plasmids of hepatitis C virus core were constructed. After identifying hepatitis C virus core protein that could stably expressed in AH109 yeast strains, we performed yeast two hybrid by mating AH109 with Y187 that transformed with liver cDNA library plasmids pACT2 and then plated on quadrople dropout (QDO) medium and assayed for alpha-gal activity. The genes of yeast colonies that could grow on QDO and had alpha-gal activity were sequenced.</p><p><b>RESULTS</b>Among the 30 positive colonies, we blasted the gene of the sixth colony; we coined human hepatitis C virus binding protein 6(Hu Hcbp6) with Genbank, realized that the Hu Hcbp6 shares as much as 98% homology with two cDNA without knowing functions. We have proved that Hu Hcbp6 could interact with hepatitis C virus core protein.</p><p><b>CONCLUSIONS</b>Hepatitis C virus core binding protein (Hu Hcbp 6 Genbank number: AY032594) was successfully cloned and identified. The study partly paved the way for investigating physiological function of the Hu Hcbp6.</p>

Preparation of human single chain Fv antibody specifically against hepatitis C virus E2 antigen / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify human single chain Fv antibody (ScFv) against hepatitis C viral E2 antigen and its value clinically.</p><p><b>METHODS</b>The recombinant phages were panned by E2 antigen which was coated in a microtiter plate. After five rounds of biopanning, 56 phage clones were identified specific to E2 antigen. The affinity and specificity of ScFv were evaluated by ELISA and immunohistochemistry, respectively.</p><p><b>RESULTS</b>The data of E2-ScFv DNA digestion and DNA sequencing showed that the ScFv gene was composed of 750bp. ELISA and immunohistochemistry demonstrated that the human single chain Fv antibody against HCV E2 antigen had a specific combination character with hepatitis C virus E2 antigen.</p><p><b>CONCLUSIONS</b>ScFv, having a sutestantial affinity and specificity and being easy to prepare, is valuable in the detection of HCV E2 antigen.</p>