Objective To evaluate the reliability of colony PCR in identifying the recombinant clones selected from phage antibody libraries. Methods The digested pComb3 vector and Lc fragments, Lc library plasmid, and Fd fragments were ligated successively. The ligation product was transformed into Escherichia Coli strain XL 1-blue bacilli by electroporation and thus murine Fab phage antibody library was constructed.The transformed recombinant clones selected randomly from libraries were identified simultaneously by colony PCR, plasmid PCR and restriction enzyme digestion. The identification consistency was analyzed. The interference was excluded by control PCR using the relevant constituents as template. Results The educed library content of Lc library was 1.175?10 6 CFU and the content of Fab antibody library was 1.02?10 6 CFU. Different recombinant percentages were obtained through three different identification methods (the Lc positive recombinant percentages by colony PCR, plasmid PCR and enzyme digestion were 100%, 78% and 78%, respectively; the Fd positive recombinant percentages by three methods were 90%, 66% and 66%, respectively; the dual positive recombinant (Fd and Lc insert simultaneously) percentages by three methods were 90%, 50% and 50%, respectively). A high frequency of false-positives appeared in colony PCR identification. Nonspecific amplification of control PCR was presumably induced by some intracellular components in XL 1-blue bacilli. Conclusion The identification of the recombinant clones selected from phage antibody libraries by colony PCR remains ambiguous. So it is our assertion that the traditional identification methods such as plasmid PCR or enzyme digestion are more accurate and will less false positive results.

Objective To construct human-mouse hybrid Fab phage antibody libraries and to select target Fab genes. Methods With Fd repertoire genes of PBMC from hepatoma patients and the chemeric light chain gene of cFab-HAb18, two hybrid Fab phage antibody libraries(pComb3 and pComb3X) were constructed. The GST fusion and non-fusion HAb18GE were used as antigens to select the target antibodies with the optimized strategies of amplifying and screening. Results When pComb3 displaying library was cultivated at 37℃, the target fragments could not be cut out with the restriction endonucleases from most clones of the library, which was an indication of “recombinant-deletion”. But no “recombinant-deletion” was found in pComb3X displaying library. Because of the serious cross-reaction, the phage-display Fab ELISA could not be used for the selection of positive clones. But positive clones could be identified in the supernatant of lysate with ELISA afterbeing induced by IPTG. Conclusion Through the construction of HuMFab phage antibody libraries and the optimization of screening strategies, we get the desired HuMFab genes for subsequent research.

Virtual experiments teaching has been characterized by openness, interactivity and re-source sharing. It efficiently improves the experiments teaching effects and promotes the teaching reform. At present the virtual experiment systems used by domestic universities can realize simulation of the ex-perimental principle, apparatus, object, operation and data. In the virtual experiment system students deepen the understanding of the experiments through foregrounding and the networked virtual experiment manage-ment effectively improves the effects of experiments teaching through behind-the-scenes action.

Objective:To construct a gene of ScFv against human HAb18G molecule and secretively express it in E coli Methods:The V H and V L genes cloned from McAb HAb18 hybridoma cell were ligated with a flexible linker(Gly4Ser) 3 to construct ScFv gene Then corresponding restriction endonuclease digestion site was introduced into 5′and 3′ end of ScFv gene Finally, it was cloned into expression vector pCANTAB 5His and expressed in E coli HB2151 Expression proteins were purified by affinity chromatography and detected by SDS PAGE electrophoresis?Western blot and ELISA Results:Restriction endonuclease digestion and DNA sequencing proved that ScFv gene was correctly cloned into expression vector SDS PAGE electrophoresis and Western blot analysis showed that ScFv was successfully expressed in E coli HB2151 The relative molecular mass (Mr) of the expression products was 31 kD, according with its predicted Mr value And ELISA assured that expression products had antigen specific binding activity Conclusion:The successful expression of anti HAb18G ScFv gene in E coli laid a solid foundation for its further application in diagnosis and therapy of human hepatoma.

Objective: To clone Fd and light chain genes of monoclonal antibody HAb18 against human hepatoma and verify their accuracy and liability. Methods: Total RNA was extracted from hybridoma cell line secreting MAb HAb18, and Fd and light chain genes were amplified by RT-PCR. After PCR products were ligated into pMD18T vector, positive clones were screened and DNA sequences were tested and analysed by relative softwares. Then, light chain and Fd genes were sequential cloned into phage display vector pComb3. After recombinant vector was transformed into E.coli XL1-blue, recombinant vector was rescued by helper phage M13K07 and the specificity of phages to antigen was detected by indirect ELISA. Results: The size of amplified Fd and light chain genes was separately 665 bp and 668 bp. The results of sequence analysis showed that both VL and VH contained 2 characteristic cystines and CH1 was IgG1 classes and CL was ?. ELISA result identified that expressed Fab antibody could specially bind to corresponding antigen. Conclusion: Fd and light chain genes of MAb HAb18 were successfully cloned, which lay a good foundation for constructing a diversity of engineering antibody.

Objective: To select anti-HAb18G (hepatoma associated antigen) human Fd fragments with guided selection of L chain of chimeric Fab-HAb18. Methods: The human Fd repertories genes were amplified by RT-PCR from PBMC of hepatoma patients, and cloned into the vector pComb3X with chimeric L chain to construct the human-mouse hybrid Fab phage library. HAblSGE, extracellular region of HAblSG, was used as antigen to screen. The positive clones was obtained by ELISA and FCM with p Ⅲ-fusion Fab antibody. The DNA sequences were analyzed. Results: A human-mouse Fab antibody library were constructed with 2?107 PFU. After 6 rounds panning, 7 positive clones were obtained with ELISA and FCM. And sequences of 2 clones with better affinity were same. The CHI belonged to the IgG2 class as the parent Fd, and the variable region belonged to VH3 family. Conclusions: Through the construction of the HuMFab phage antibody library and chemeric L chain-guided selection, we get the available human Fd fragments for subsequent research.

Objective To study the relationship between the clinical imaging manifestations and the judgement of benign and malignant of solitary pulmonary nodules (SPN) .Methods A retrospective analysis of 146 patients with SPN which confirmed by pathological diagnosis .According to SPN diameter all SPN were divided into three groups ,the number of SPN which diameter smal-ler than 0 .8 cm were 16 case ,> 0 .8 - 1 .5 cm were 41 cases ,> 1 .5 - 3 .0 cm were 89 cases .Collect patients′ age ,sex ,smoking his-tory ,clinical symptoms and imaging data ,and analyze its relationship with pathological results .Results All the 146 patients were pathological diagnosis ,benign in 52 cases (35 .6% ) ,malignant in 94 cases (64 .4% ) .As the diameter increases ,the malignant rate increased .There was statistical significance in benign and malignant lesions with smoking history and clinical symptoms (P< 0 . 05) .Whether SPN boundary is clear ,have lobulation ,burr ,pleural indentation syndrome and vessel convergence in benign and ma-lignant lesions were statistically significant (P< 0 .05) .Conclusion The feature size and imaging performance of SPN has important reference value for the judgement of benign and malignant .The positive intervention to SPN have great significance on improve sur -vival rate of lung cancer .

Objective To summarize clinical experience in removal of residual metallic foreign body in the soft tissue.Methods Clinical data of 742 cases with residual metallic foreign body in the soft tissue were analyzed.All the patients were forward from other hospitals with failed removal of the foreign body.Second surgery was performed in our hospital by grasping forceps using 3D-locator under the guidance of the C-shaped arm X-ray machine.Results Foreign body,such as scrap-iron,broken needle,nails,wine and so on located at different regions of soft tissues including neck,chest and abdomen,pelvis,and the four limbs were all successfully taken out.Removal rate of the foreign body was 100％.No complications such as bleeding,infection and nerve damage was occurred.The mean time of the procedures and radiation exposure for the removal surgery was 5 minutes.Conclusion Using 3D-locator and grasping forceps under the guidance of the X-ray,the residual metallic foreign bodies can be removed safely and efficiently.

Objective To study the consistency in the diagnosis of preoperative TNM rectal cancer staging using high resolution magnetic resonance imaging (MRI) and transrectal ultrasound (TRUS) combined with carcinoembryonic antigen (CEA) and postoperative pathological TNM.Methods 156 cases pathologically proven were retrospectively analyzed and divided into 4 groups including preoperative MRI group (39 cases),TRUS group (39 cases),MRI and TRUS group (39 cases),MRI and TRUS combined with CEA group (39 cases).The differences between preoperative T,N staging and postoperative pathologic T,N staging were analyzed.Results There were statistically significant differences in the diagnosis of preoperative T and postoperative pathological T in 4 groups (T: Kappa =0.685,P =0.000; N: Kappa =0.544,P =0.000),but there were no significant differences in preoperative N and postoperative pathological N staging in preoperative MRI group,TRUS group,MRI and TRUS group (Kappa =0.142,P =0.329; Kappa =0.154,P =0.645; Kappa =0.154,P=0.229),and significant difference was observed in MRI and TRUS combined with CEA group (Kappa =0.544,P =0.000).There were no significant differences in the accuracy of T staging among the 4 groups (x2 =0.326,P =0.574; x2 =0.562,P =0.719; x2 =0.287,P =0.986),but significant difference in the accuracy of N staging were showed among the 4 groups (x2 =4.643,P =0.026; x2 =6.643,P =0.026; x2 =5.243,P =0.019).Conclusion Preoperative evaluation by the MRI add TRUS combined with CEA can improve the accuracy of preoperative staging,which can provide more reliable basis for decision-making and improve the coincidence rate of operative procedures in line with the estimate.It also provides the basis fur the accurate preoperative diagnosis and individualized treatment.

Objective To investigate the effects of different concentration and inhalation duration time of isoflurane on cognitive performance and the expression of GABAR1 and NMDAR2B in aged SD rat cerebral temporal lopes.Methods Aged male SD rats (9 months) were randomly divided into control group (n=10) and test group (n=80).The control group received air at room tempreture.Test groups were divided into four groups: group S1 (1.5%-2 h),group S2 (2.5%-2 h),group S3 (1.5%-4 h),group S4 (2.5%-4 h)according to isoflurane concentration and inhalation duration time.Every group was equally divided into two groups and Morris water maze test was performed day 1 and day 7 after isoflurane inhalation.Then the right temporal lobe was gathered and the mRNA transcription and protein expression of GABAR1 and NMDAR2B were detected by RT-PCR and Immunofluorescence technique.Results One day after isoflurane inhalation, accompanied with increased isoflurane concentration and inhalation duration, the spatial memory ability of every test group decreased continually, and the mRNA transcription and protein expression of GABAR1 increased and the mRNA transcription and protein expression of NMDAR2B decreased compared with control group (P<0.01).Seven days after isoflurane inhalation, the spatial memory ability of group S4 decreased, the mRNA transcription and protein expression of both GABAR1 increased, the mRNA transcription and protein expression of NMDAR2B decreased compared with control group and the other test groups (P<0.01).There was no significant difference between the control group and groups S1, S2, S3.Conclusion Continuous inhalation of isoflurane has great effects on spatial memory ability.And impaired spatial memory by isoflurane inhalation of high concentration with long duration is present in a long time.Thoses are related with the mRNA transcription and protein expressions of GABAR1 and NMDAR2B in cerebral temporal lope.