Abstract:Abstract: Bacterial sRNAs are a class of non-coding RNAs with 40-500 nucleotides in length. Most of them function as posttranscriptional regulation of gene expression through binding to the translation initiation region of their target mRNAs. In view that prediction of sRNAs and their targets provides support for experimental identification, some prediction methods have been developed for both of them in recent years. In this review, we firstly gave an overview of methods for prediction of sRNA genes, which are classified into three categories, namely, comparative genomics-based, transcription units-based and machine learning-based prediction methods. Secondly, the methods for sRNA target prediction are classified into two types, which are sequence alignment-based method and prediction of RNA secondary structure-based method, respectively. Finally, the principles, advantages and limitations of each kind of method are discussed, and perspectives for prediction methods of sRNA and their targets is pointed out.

Abstract:Trehalose synthase inverts maltose into trehalose, and plays a very important role in trehalose industrial production. Here we reviewed the recent progress on studies of molecular biology including the cloning of trehalose synthase gene, gene engineering application, structure and catalytic mechanism and the role of the enzyme in vivo.

Abstract:2008年度，国家自然科学基金项目申请实行了新的“申请代码”，使其能够体现科学基金不断适应变化了的基础研究状况，同时也体现基金资助项目更加侧重基础、更加侧重前沿的思想。本文对申请代码的修订、2008年度微生物学学科申请与资助项目情况进行了总结和分析,希望能为科研人员在2009年及今后申请国家自然科学基金项目时提供参考。

Abstract:[Objective] To study the morphological changes of Cytophaga hutchinsonii cell during its life circle. [Methods] Cytophaga hutchinsonii cell was observed under light microscope, fluorescence microscope and scanning electron microscope. The nucleoids in the cell were stained with fluorescent dye Hoechst33342 and examined by fluorescence microscopy. [Results] We discovered that under starvation conditions, the long, flexible rod cell of Cytophaga hutchinsonii would bend and turn into circular cell. The circular cell failed to produce carboxymethyl cellulase. Some of the circular cells might further wind around and turn into tiny spherical cells. The tiny spherical cell similar to the microcyst of sporocytophaga could germ into long flexible rods again under certain circumstances. When growing cultures to logarithmic phase of cell growth, Cytophaga hutchinsonii cell with three nucleoids in it was occasionally observed, which indicated that the two strands of DNA might act differently in the initiation of DNA replication. [Conclusion] This is the first detailed description of the formation process of circular cell and tiny spherical cell in the life circle of Cytophaga hutchinsonii. The result will help to further reveal the relation between morphologic change and cellulose degradation ability of the strain.

Abstract:[Objective] Construction of eag deletion mutant of Bacillus anthracis vaccine strain A16R. [Methods] To study the function of the gene eag of Bacillus anthracis vaccine strain A16R, according to the sequence of Bacillus anthracis Ames strain, we designed primers and constructed a recombinant plasmid by the spectinomycin resistance cassette, upstream homologous fragment and downstream homologous fragment of eag cloned in tandem in pKSV7. We introduced the recombinant into A16R by electroporation and screened the mutant using the principle of homologous recombination. We checked the mutant using the PCR and proteomics. [Results] We constructed the recombinant plasmid successfully and got the eag deletion mutant. PCR results showed the gene eag was deleted; SDS PAGE showed evident differences between prime strain and mutant strain. Two-dimensional gel electrophoresis results displayed three protein points identified as EA1 by mass chromatographic analysis presented in prime strain had absented from the mutant strain. [Conclusion] We constructed eag deletion mutant of Bacillus anthracis vaccine strain A16R?. This research will be helpful to study the functions of eag gene and the functions of the important genes of Bacillus anthracis.

Abstract:[Objective] To better understand the mechanisms of cyclic di-GMP signaling in Xanthomonas oryzae pv. oryzae (Xoo), the casual pathogen of bacterial blight of rice, molecular identification of Clpxoo. [Methods] A putative signal receptor protein was performed through gene cloning, sequencing and deletion analysis. [Results] Sequence analysis showed Clpxoo was a homologue of Crp and Vfr, the cAMP receptor proteins in Escherichia coli and Pseudomonas aeruginosa respectively, which had the cNMP-binding domains (CAP_ED) at N terminal and the DNA-binding domains (HTH_CRP) at C terminal and is highly conserved in the plant-pathogenic Xanthomonas spp. We constructed △clpxoo through a double crossover recombination and validated by PCR assay, △clpxoo displayed the reduced motility and extracellular polysaccharide (EPS) production, and increased sensitive to H2O2 toxicity compared with PXO99A. All these phenotype changes could be partly restored through complementation of mutants by introducing clpxoo. Moreover, we observed no significant changes in production of extracellular cellulase and xylanase in vitro, biofilm formation and induction of hypersensitive response (HR) on non-host tobacco in △clpxoo compared to PXO99A. [Conclusion] Therefore, Clpxoo played a role as one of the global regulator in regulation of flagellar motility, EPS production and H2O2 resistance.

Abstract:[Objective] Wes identified a neomycin-resistant mutant with reduced membrane-bound H+-ATPase activity from L. delbrueckii subsp. bulgaricus originated from the traditional dairy product to develop a yoghurt starter culture with low post-acidification capacity. [Methods] API 50 CH identification system and 16s rDNA sequence analysis were applied to identify the strain isolated from the indigenous yoghurt. Neomycin was used to screen spontaneous neomycin-resistant mutants. H+-ATPase activity and metabolic dynamics were evaluated between the parent strain and mutants. [Results] One strain was identified as L. delbrueckii subsp. bulgaricus by API 50 CH identification system and 16s rDNA sequence analysis, coded as KLDS 1.9201. Two mutant strains were mutated from KLDS 1.9201 and coded as KLDS 1.9201-1, KLDS 1.9201-4. Compared with the parent strain, the H+-ATPase activity of the mutants KLDS 1.9201-1 and KLDS 1.9201-4 decreased by 46% and 60% respectively. After cultured in MRS broth for 24 h, metabolic efficiency of the initial glucose for the parent strain was 65%, the mutants were 41% and 31%, the lactic acid concentration in the culture for the parent strain was 26g/L, the mutants were 18g/L and 15g/L respectively. The cell density of the mutants was lower than that of the parent strain. [Conclusion] The mutants of L. delbrueckii subsp. bulgaricus with reduced membrane-bound H+-ATPase activity had low growth and acid production, which could be applied to develop yoghurt starter culture with lower post-acidification.

Abstract:[Objective] We obtained a new mutant with higher growth rate and high capability of producing arachidonic acid after protoplast ultraviolet mutation. [Methods] Using dry weight of biomass, microbial total lipid and total arachidonic acid production as evaluation index, we determined time of ultraviolet irradiation by single factor experiment and measured the content of the arachidonic acid by gas chromatography. [Results] When the power of viltalight lamp was set at 20W, exposure distance at 30cm and exposure time at 80s, lethality of spores of Mortierella isabellina was 76.4%. [Conclusion] After ultraviolet mutation and repeatedly screening, a mutat of Mortierella isabellina YZ-124 whose arachidonic acid concentration in biomass was 5.76 times of the control strains was obtained.

Abstract:[Objective] To analyze the co-production of thrombolytic enzyme and γ-polyglutamic acid by liquid-culture of Bacillus subtilis SBS. [Methods] We used Bacillus subtilis SBS to produce thrombolytic enzyme (BTE) and γ-polyglutamic acid (γ-PGA) by liquid fermentation. We conducted orthogonal experiments analysis between carbon source and nitrogen source. Then through Fibrin-SDS-PAGE, UV spectrum, Infra-Red spectrum and high performance liquid chromatography (HPLC), we identified the production. [Results] We synthesized γ-PGA in the medium without the addition of L-glutamic acid. Bacillus subtilis SBS was a L-glutamic acid-independent bacterium. The suitable carbon and nitrogen sources for the synthesis of thrombolytic enzyme (BTE) were soluble starch and soybean peptone. However, the suitable carbon and nitrogen sources for the synthesis of γ-PGA were sucrose and NH4Cl. [Conclusion] The yields of both products could close to the levels of the single synthesis when the concentrations of sucrose, soybean peptone and NH4Cl were 10, 20, 8 g/L respectively, the activity of BTE reached 265±25 IU/mL, and the concentration of γ-PGA reached 1.183±0.015 g/L.

Abstract:[Objective] Staphylococcus aureus is a member of Gram positive bacteria, but is also one of common pathogens that are most difficult to deal with. It infects human skin, soft tissue, mucous membrane, bone and joint, especially in the nosocomial environment. Because studies on Staphylococcus aureus before were largely based on a single gene or protein, it is necessary to study this organism from the whole genome. [Methods] We used bioinformatics methods including five computational methods (phylogenetic profile, gene neighbor method, operon method, gene fusion method, interolog) to predict the protein interaction network of Staphylococcus aureus. [Results] We constructed the protein interaction network of Staphylococcus aureus and studied its function. [Conclusion] Through the network analysis, we found that the protein interaction network of Staphylococcus aureus was subject to scale-free property and a number of very important proteins, such as SA0939, SA0868, rplD. Through the analysis of the important cell wall synthesis and signal transduction and regulation local networks, we also found some very important proteins. Such information will help us better understand pathogenic mechanism and develop new drug targets of Staphylococcus aureus.

Abstract:[Objective] To know the activities of dissolving Ca3(PO4)2 and FePO4 of phosphate-solubilizing bacteria (PSB) isolated from acidic soil in Hainan province in China and to screen PSB with high and stable activity (PSBHS). [Methods] Soil samples of 21 plant species were isolated with nutrient agar, crystal violet-nutrient agar, and yeast extract-mantol agar with dilution-plate method. Bacterial representative of the predominant morphologically distinct colonies present on plates were selected randomly and purified on minimal nutrient agar. First screening was done on solid medium with tricalcium phosphate (Ca-P) as the sole P source, and after 5 days of cultivation the colonies with phosphate-solubilizing zones were selected for further screening. Secondary screening was conducted by cultivating the selected colonies in Ca-P liquid medium for 6 days at 32℃ with shaking of 200 r/min, and PSB capable of solubilizing phosphorus up to more than 200 mg/L were selected. The third screening—6-day shaking cultivation in Ca-P liquid media—was conducted after 4 successive cycles of transfer-culture plus 15 d-storage at 4℃. PSB capable of solubilizing phosphorus up to more than 200 mg/L was selected and termed as PSBHS. Then PSBHS were cultivated in FePO4 liquid media for 6 days, and the activity of dissolved phosphorus was determined. The 16S rDNA gene sequencing and blast search against the public database were used to identified PSBHS. [Results] In total 363 bacterial representatives were isolated from soil samples, and the following three screenings reduced the number of representatives to 126, 45, and 14 respectively. The amounts of dissolved phosphorus by 14 PSBHS after 6 days of cultivation in Ca-P liquid media were up to 201.0 mg/L~623.3 mg/L, the pH values of media decreased to 3.82~4.34 from initial 7.0, and the final pH values were found to be significantly negatively correlated to the amounts of dissolved phosphorus. After 6-day of cultivation in FePO4 liquid media, only a small amount of phosphorus (1.6 mg/L ~34.2 mg/L) were solubilized by PSBHS, and the final pH values of media decreased to 2.87~5.67— also significantly negatively correlated to the amounts of dissolved phosphorus. The 16S rDNA gene sequencing analysis identified 6 PSBHS to be Acinetobacter, 3 to be Pseudomonas, 3 to be Serratia, 2 to be Enterobacter.

Abstract:[Objective] To investigate the feasibility of using attenuated Salmonella typhimurium as carrier for oral immunization of TGEV DNA vaccine. [Methods] 2.1 Kb fragments of the TGVE SC-H strain S gene that encompasses all the four major antigenic domains was amplified by RT-PCR and cloned into eukaryotic expression vector pVAX1. The recombinant plasmid pVAX-S was transfected into COS7 cells and the expression of recombinant plasmids was identified by indirect immunofluorscence assay. Then pVAX-S was transformed by electroporation into attenuated Salmonella typhimurium SL7207, The recombinant was screened and designated as SL7207(pVAX-S). Mouse peritoneal macrophages were infected with SL7207(pVAX-S), the transcription and expression of S gene were detected by RT-PCR and indirect immunofluorscence . BALB/c mouse were inoculated orally with SL7207(pVAX-S) at dosage of 5×108、1×109、2×109CFU for safety analysis. In a vaccination test, BALB/c mouse were immunized orally with recombinant bacterial at dosage of 1×109 CFU, for three times and specific serum IgG and intestinal mucosa1 IgA antibody were detected by indirect ELISA. [Results] Recombinant plasmid pVAX-S was constructed correctly and expressed in COS7 cells. The transcription and expression of S gene was detected after mouse peritoneal macrophages were infected with SL7207(pVAX-S). The recombinant bacterium was safe to mouse at dosage of 2×109CFU. Specific serum IgG and intestinal mucosa1 IgA antibody against TGEV S protein were detected in SL7207 (pVAX-S) immunized group at 2 weeks post-boosting, and there were significant difference (P<0.05) in serum IgG and most significant difference (P<0.01) in intestinal mucosa1 IgA at 2 weeks post-three immunization compared with SL7207(pVAX) control group. [Conclusion] The recombinant Salmonella carried TGEV S gene DNA vaccines had good immunogenicity and safety in mouse.

Abstract:[Objective] In recent years, manipulation of large herpesvirus genomes has been facilitated by using bacterial artificial chromosome (BAC) vectors. We have previously reported the construction of the BAC clones (HVT BACs) of herpesvirus of turkey (HVT). [Methods] With these BAC clones in hand, we manipulated the genome of HVT by utilizing Red/ET recombination system, and we were trying to develop a biologically safe live vaccine based on the HVT BACs. In this two-step approach, we first transformed the plasmid pRedET into the DH10B competent cells that carried the HVT BACs, and then added inducer L-arabinose into the cells and shifted the temperature from 30℃ to 37℃, after 45 minutes induction, we prepared the cells into competent cells,then we electroporated the linear rpsL-neo counter-selection/selection cassette flanked by the 50 bp long homology arms into the cells, RecE and RecT catalyze the homologous recombination reaction, the functional cassette was inserted into the US2 locus to be modified of HVT. Only colonies carrying the modified BAC would survive Kanamycin selection on the agar plates. The successful integration of the rpsL-neo cassette was monitored by PCR and Streptomycin selection, for the insertion of rpsL-neo cassette cells will become Streptomycin sensitive. Secondly, in the same way, we replaced the rpsL-neo cassette with the hemagglutinin(HA )gene of (HPAIV)A/Goose/Guangdong/1/96(H5N1)flanked by the same homology arms. Only colonies which lost the rpsL-neo cassette will grow on Streptomycin containing plates. [Results]Finally, we obtained many colonies of which the HA gene of the AIV was inserted into the US2 locus to be modified of HVT. And we reconstituted one recombinant virus from transfecting one of these BAC clones DNA into chick embryo fibroblasts(CEFs). [Conclusion]We achieved one rescued recombinant virus which designated as rHVT-HA3.The H5 subtype HA gene expression in this recombinant virus rHVT-HA3 was confirmed by immunofluorescence assay.

Abstract:[Objective] We cloned and expressed chicken interferon-gamma gene (chIFN-γ), and detected the bioactivity of chIFN-γ expressed in Escherichia coli (E.coli) . [Methods] The chIFN-γ mature protein gene was amplified by RT-PCR from spleen lymphocytes of chicken which were stimulated with concanavalin A (ConA) and then cloned into the prokaryotic expression vector pET-32a (+). Recombinant expression plasmid of pET-32a (+)-chIFN-γ was constructed and then transformed into the competent E. coli BL21 (DE3) cells for expression with IPTG induction. Purified soluble rchIFN-γ proteins were obtained by Ni-NTA His Bind Resin affinity chromatography and identified by SDS-PAGE gel and Western blot assay. The antiviral activity was detected by MDCK-VSV system. [Results] A 456 bp cDNA encoding chIFN-γ mature protein gene was cloned and successfully expressed in E.coli with approximate molecular weight of 31.0 kDa, which could be recognized by anti-His mAb and rabbit polyclonal antibody against chIFN-γ. The recombinant chIFN-γ proteins were expressed to form inclusion bodies with a portion of soluble protein. The soluble rchIFN-γ proteins were purified by Ni-NTA column under a native condition with the yield of 3.0 mg/L. The purified recombinant chIFN-γ (rchIFN-γ) proteins by 1:32 dilution could resist 100 TCID50 VSV virus attack. [Conclusion] The purified rchIFN-γ proteins by Ni-NTA column under a native condition had better antiviral activity, which will establish a basis for further developing new type antiviral interferon praeparatum.

Abstract:[Objective] Low temperature oxygen plasma was used to sterilize the Pseudomonas aeruginosa samples on the polyethylene terephthalate (PET) sheets in a self-designed reactor that included the discharge area, afterglow area and remote area. [Methods] Before and after plasma treatment, the cell morphology was studied by scanning electron microscopy (SEM). The cell wall or cell membrane cracking was testified to determine the content of protein by using coomassie light blue technique. Besides, double Langmuir probe and electron spin resonance spectroscopy were used to determine the temperature of electrons (ions) and the concentration of free radicals, respectively. [Results] Under the treatment time of 30 s, the germicidal effects were 4.2, 3.8 and 2.6 respectively in the three areas. SEM observations showed that the plasma activity cracks the cell wall and cell membrane, resulting in cellular content leakage. In addition, the results from electron spin resonance spectroscopy and double Langmuir electron probe showed that electrons, ions and oxygen free radicals played important roles in sterilization in the discharge area, but only oxygen free radicals acted to sterilize the bacteria in the afterglow area and the remote area. [Conclusion] The active species can be separated effectively in this reaction equipment, and we further elucidated the mechanisms of plasma sterilization in the remote plasma field.

Abstract:[Objective] To rapidly identify EV71 and CA16 simultaneously. [Methods] A multiplex real-time PCR with an internal control (IC) was developed. The specificity, sensitivity, reproducibility of the real time RT-PCR assay were estimated and more over 400 clinical samples were tested. [Results] The results showed 100% specificity for the selected panel. The assay met the sensitivity of 50% tissue culture infective dose (TCID50) per milliliter samples for CA16 and 0.1 TCID50 for EV71. Analysis with 104-0.1TCID50/mL EV71 or CA16 samples demonstrated high reproducibility with a coefficient of variation (CV) of 0.9-2.0% for EV71 and 0.9-2.3% for CA16. More than 400 clinical samples were detected by real-time PCR, The results showed that the average positive ratio for EV71 and CA16 were 46.1% and 14.2%, higher than common assay (34.5% and 12.8%). Moreover, the result statistics indicated that there were PCR inhibition in stools, rectal swabs and throat swabs specimens with inhibition ratio from 1.8% to 3.4%. The inhibition ratio of these samples showed the importance of IC when PCR was used to detect the RNA of EV71, CA16 or other enterovirus. [Conclusion] As a result of its high specificity, sensitivity and avoiding false negative results by using an internal control , the assay is suitable for rapid clinical diagnosis of EV71 and CA16.

Abstract:[Objective] To obtain new pharmaceuticals and enzymes with high activity, we studied the composition as well as antimicrobial and enzyme activities of actinomycetes in Grand Shangri-La. [Methods] Using 4 media, we isolated mesophilic and psychrophilic actinomycetes from 220 soil samples collected from areas with different altitudes in Grand Shangri-La. Twenty-five representative isolates were phylogenetically analyzed based on their 16S rRNA gene sequences. Antimicrobial activities against four bacteria and seven fungi were tested using agar well diffusion method. Genes encoding typeⅠandⅡ polyketide synthases (PKSⅠ, PKSⅡ), nonribosomal peptide synthase (NRPS) and polyene cytochrome P450 hydroxylase (CYP) were screened by PCR. Furthermore, several enzyme activities of psychrophilic actinomycetes were examined. [Results] The 25 representative strains belonged to 6 suborders, 12 families and 15 genera of the order Actinomycetals. For NRPS and CYP genes screening, positive strains were 14 and 11, respectively. Among the 111 actinomycetes isolated under low-temperature conditions, 88% were psychrotroph strains, 12% were psychrophilic actinomycetes, and most of them utilized gelatin, cellulose and chitin. [Conclusion] Actinomycetes diversity isrich in Grand Shangri-la, and has the potential for conservation and utilization of actinomycetes resources.

Abstract:[Objective] To survey the distribution of vip3A-type genes from isolates of Bacillus thuringiensis from China and clone novel vip3A genes encoded Vip3A proteins with high insecticidal activity against Lepidopteran insect larvae. [Methods] We applied a PCR-RFLP method to identify vip3A-type genes from 171 isolates and cloned novel vip3Aa genes. [Results] The vip3A-type genes appeared in 63 of 117 B. thuringiensis isolates. We cloned two novel vip3Aa genes from isolates of TF9 and Bt16. Then, we subcloned vip3Aa26 and vip3Aa27 into vector pQE30 and transformed into Escherichi coli M15, respectively. The results of SDS-PAGE and Western blot analyses showed that a 88 kDa peptide was expressed in E.coli M15 with 1mmol/L of IPTG induction at 37 centigrade, respectively. The International Nomenclature Committee of Bt nominated these two genes as the novel vip genes of vip3Aa26 and vip3Aa27, respectively. The bioassay results indicated that the Vip3Aa27 proteins was highly toxic to Trichoplusia ni,Spodoptera exigua and Helicoverpa armigera larvae and the LC50 values were 0.125 μg/mL, 0.238 μg/mL and 9.238 μg/mL, respectively. However, the Vip3Aa26 protein only possessed toxicity to T.ni larvae. [Conclusions] The novel Vip3Aa27 protein had higher activity to Lepidopteran insect larvae compared with that for Vip3Aa26 protein. The results demonstrated that some amino acids changes had remarkable effect on the insecticidal activity.

Abstract:[Objective] To study the inhibition of human gastric cancer cell BGC823 by exopolysaccharide (B. EPS) extracted from Bifidobacterium bifidum and the effect on the activity of telomerase rate-limiting factor hTERT. [Methods] The B. EPS was divided into three groups by multiplication concentration level which effected on human gastric cancer cell BGC823 in vitro, the inhibition rate of gastric cancer cell BGC-823, the morphology changes of cells and the apoptosis in the initial stage of cell was observed respectively by MTT, inverted fluorescent microscope and the Annexin V-FITC/PI flow cytometry. In addition, the effect of B.EPS on cell telomerase rate-limiting factor hTERT mRNA was tested by RT-PCR; the changes in the concentration of calcium ions inside the cytoplasm were detected by spectrofluorometer. [Results] B.EPS inhibited the growth of gastric cancer cell BGC-823 (P<0.05), the inhibition rate increased and the concentration of B.EPS showed dose-time response relation. The expression of cell telomerase rate-limiting factor hTERT mRNA decrease after effected by B.EPS (P<0.05), and showed dose-effect relation. The concentration of calcium ion in cytoplasm was higher than the control apparently (P<0.05). [Conclusion] The apoptosis mechanism of human gastric cancer cell BGC823 induced by B.EPS may be relevant with the expression of hTERT mRNA and the concentration of calcium ions in cytoplasm.

Abstract:?Objective? To establish stable expressing system of Borna disease virus (BDV) phosphoprotein in PC-12 cells, and then study its influence on cell proliferation of PC-12 cells. ?Method? An expression plasmid with green fluorescence protein was cloned and identified to express BDV phosphoprotein. Cultured PC-12 cell was transfected with the recombinant plasmid by positive ion lipidsome method. Fluorescence microscopy was used to detect the expression of phosphoprotein in PC-12 cells, then G418 was added into cell culture medium to kill these cells without recombinant plasmid. We performed reverse transcriptase polymerase chain reaction (RT-PCR) in the 10th generation of treated cells to examine the expression of BDV phosphoprotein. The proliferation of treated cells and control cells was examined by methyl thiazolyl tetrazolium assay (MTT). ?Result? The recombinant plasmid was confirmed to be able to express BDV phosphoprotein and green fluorescence protein by both fluorescence and RT-PCR. BDV phosphoprotein expressed in PC-12 cell inhibited cell proliferation. ?Conclusion? We established a stable expressing system of BDV phosphoprotein in PC-12 cell. This cell model can be used to study the effect of BDV phosphoprotein on the centre nervous system without exposure to live virus.

Abstract:[Objective] Escherichia coli is the best characterized system in every aspect, and it has been the most widely used host for the production of recombinant proteins. Furthermore, the high cell density culture (HCDC) has allowed production of various proteins with high yield and high productivities. Automation and miniaturization are key issues of high-throughput research projects in the post-genomic era. [Methods] We applied a new culture-complex auto-inducing media (CAI) for heterogenous protein expression in E. coli. The CAI is in accord with automation and miniaturization. To test expression efficiency in the CAI, we constructed seven different plasmids named p-1、p-2、p-3、p-4、p-5、p-6 and p-7. These plasmids were transformed into E. coli BL21 (DE3), then expressed in both LB and CAI. [Results] The expression levels of seven fusion proteins in CAI were four times greater than those in LB. To improve the expression level even more, we analyzed the composition of the CAI and optimized the culture. Through a series of changes we formed a new optimized culture (CAI-4). Comparing the expression levels of there fusion proteins (P-1、P-2、P-3) in CA, the expression levels of fusion proteins in CAI-4 were twice greater than the control.

Abstract:[Objective] To screen and identify anti- Aflatoxin B1 single chain antibodies (scFv) from Tomlinson (I) library. [Methods] The phages absorbed on the ELISA plate were eluted by four methods including glycine buffer elution, trypsin elution, AFB1 competitive elution and competitive elution following trypsin treatment. The phage positive clones were transformed into Escherichia coli HB2151 and soluble scFv protein was expressed with the induction of Isopropyl β-D-1-Thiogalavtopy ranoside (IPTG). The scFv was identified by ELISA and DNA sequence. [Results] Comparing the four methods, we found that the most efficient way to get positive clones was competitive elution following trypsin treatment. Obtainning two positive clones that could bind specifically with free AFB1, which their relative affinity were 0.4 μg/mL and 0.7 μg/mL, respectively. DNA sequencing results showed that the scFv belonged to human immunoglobulin variable region. [Conclusion] The specific human scFv could be obtained with phage antibody library, and our method provided an alternative for screening recombinant antibody against anti-hapten.