Chinese Medicine Intervention on Autophagy in Lung Cancer from Theory of Healthy Qi Deficiency and Pathogenic Qi Stagnation： A Review / 中国实验方剂学杂志

Autophagy， a mechanism of cell self-protection and self-renewal， is associated with the occurrence and development of lung cancer. Favorable autophagy can slow down the progression of lung cancer， while unfavorable autophagy can promote the progression. Therefore， regulating the level of autophagy is of great significance in the treatment of lung cancer. Healthy Qi deficiency and pathogenic Qi stagnation is an extension of the theory of deficiency and Qi stagnation proposed by the Academician WANG Yongyan. It refers to the pathological process that the abnormal body fluid metabolism caused by Qi deficiency of lung， spleen， and kidney results in phlegm and blood stasis. Lung cancer has the root cause of Qi deficiency of lung， spleen， and kidney and the syndrome of phlegm and blood stasis. The autophagy in lung cancer is interconnected with healthy Qi deficiency and pathogenic Qi stagnation. The Qi deficiency of lung， spleen， and kidney is the key factor for the weakening of favorable autophagy in lung cancer， which inhibits the apoptosis of tumor cells and leads to the accumulation of harmful substances. Phlegm and blood stasis is a direct factor enhancing the unfavorable autophagy in lung cancer， which promotes the autophagic death of normal cells， weakens the immunosuppressive effect of immune cells on tumor cells， and leads to the proliferation and migration of tumor cells. The combination of healthy Qi deficiency and pathogenic Qi stagnation results in the development of autophagy in an unfavorable direction and finally leads to the continuous progression of lung cancer. Therefore， the traditional Chinese medicine （TCM） treatment of lung cancer should follow the principle of reinforcing healthy Qi and expelling pathogenic Qi， removing phlegm and resolving stasis， so as to enhance favorable autophagy while inhibiting unfavorable autophagy. Such therapy can inhibit the proliferation and migration of tumor cells and promote the remission of lung cancer. According to the existing literature， Chinese medicine monomers are mainly used to treat lung cancer by regulating autophagy. The Chinese medicine intervention of autophagy in lung cancer mainly aims to promote the activation of autophagy. This may be because the favorable autophagy weakening caused by the Qi deficiency of lung， spleen， and kidney is the fundamental reason for the development of lung cancer.

Association of Apolipoprotein E Polymorphisms with White Matter Lesions and Brain Atrophy

Objective@#Apolipoprotein E (ApoE) is mainly synthesized in the liver. So far, it is unknown the relationship among APOE gene polymorphisms and WML, brain atrophy. Therefore, the aim of the study was to assess the associations of APOE gene polymorphisms in patients with WML and brain atrophy. @*Methods@#A total of 58 patients with WML, 128 patients with brain atrophy, 112 patients with co-occurrence of WML and brain atrophy and 95 healthy elderly volunteers were recruited from Renmin Hospital of WuHan University. @*Results@#Allele E3 was the most common allele. The alleles E2 had significantly higher levels of ApoB and lower age in WML group. The alleles E2 was associated with the lower level of ApoB, LDL-Ch, TCh, and sdLDL in co-occurrence group. The E3/E3 genotype has higher level of sdLDL, but lower age and female frequency in WML. The E3/E4 genotype had higher level of TG, but lower age in WML. Gender, Age, E2, Hyperhomocysteinemia and UA were also significantly associated with disease progression. @*Conclusion@#This study found that clinical data, lipids and metabolic complications were closely related to ApoE genotypes and alleles, and also disease progression and type.

Role of basophils in the imbalance of Th1/Th2 response in collagen-induced arthritis in mice / 中国比较医学杂志

Objective To investigate the role of basophils in the imbalance of Th 1/Th2 response in mouse models of collagen-induced arthritis(CIA).Methods 4-6-weeks old C57/BL6 mice were immunized with collagen at multiple points on the back and foot twice (0 and 3 weeks) to establish a mouse model of collagen-induced arthritis.Blood samples were collected before the first immunization and 1, 3, 6 and 9 weeks after immunization , and cells from lymph nodes were collected.Flow cytometry and ELISA were employed to detect the levels of basophils and IL-4, and the joint swelling was scored.Results Mouse model of CIA was successful established .The ratio of IL-4/IFN-γof the CIA group was significantly lower than that in the mice before CIA modeling and the control group , indicating a Th2-dominant response .At the same time, the peripheral basophils counting and percentage of IL-4 positive basophils of the CIA group were significantly higher than those of the control group .While, the IL-4/IFN-γratio of the CIA group was significantly higher than that of the control group , indicating a Th1-dominant response .The peripheral basophils counting of the CIA group was slightly lower than that of the control group .Conclusion Basophils may participate in the development of CIA in mouse models through affecting the imbalance of Th 1/Th2 response.

Hepatitis B virus X protein regulates the mEZH2 promoter via the E2F1-binding site in AML12 cells / 癌症

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma (HCC) tissues and associated with hepatocarcinogenesis. However, the exact mechanism of EZH2 up-regulation in HCC has not been determined. In this study, we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein (HBx) in regulating the expression of mEZH2. Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner. To further investigate the mechanism of mEZH2 overexpression, the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid. The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid, and deleting the -486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%. The -486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found. Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells. These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor, thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

Cloning NS1 gene of H5N1 avian influenza virus and apoptosis induced by it in human pulmonary carcinoma cell line A549 / 病毒学报

The NS1 gene of the H5N1 subtype avian influenza virus was amplified by RT-PCR, and the am-plified product was cloned into the eukaryotic expression vector pCMV-Myc, then it was transfected into A549 cells. After 48 h, the expression of NS1 was detected by Western blot. Fluorescence and electron microscopy and flow cytometry showed that the NS1 gene of H5N1 avian influenza virus could induce apop-tosis in human pulmonary carcinoma cell line A549.

Enhancement of exogenous gene expression by artificial transcription factor in CHO cells / 生物工程学报

Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA-binding domain and four tandem repeats of the 12-aa peptide (DALDDFDLDMLG) of the herpesvirus as the activation domain, an artificial transcription factor, GVP4,was constructed via the linkage of the nuclear localization signal sequence of SV40. And then, GVP4 was cloned into expression vector pcDNA3 . 1/Hygro ( + ) . Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1 ( + ) that separately harbored EGFP cDNA and t-PA cDNA.The CHO cells were then co-transfected with GVP4 expression vector and EGFP or t-PA expression vector. The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t-PA in the supernatant. GVP4 showed positive effect on the enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor. And, CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression, which resulted in 2-3-fold increase in both EGFP and t-PA expressions. These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.

The targeting study of human serum albumin gene / 生物工程学报

To generate transgenic porcine which expresses human serum albumin (HSA), the HSA gene targeting vector was constructed with HSA cDNA as the gene of interestand partial porcine serum albumin (PSA) gene as homologous arms which respectively were 7.2 kb 5' regulation sequence and 2.8 kb genomic sequence from the first intron to the fourth intron. The resistant gene neo was inserted into intron 1 and tk was ligated to the 3' end of the construct. Linearized targeting construct DNA was introduced into the fibroblast cells obtained from porcine fetus by electroporation. The positive-negative selection was performed and survival clones were screened by PCR and Southern blot. Three colonies with correct homologous recombination were obtained. Our results set a good basis for the establishment of transgenic porcine by gene target and nuclear transfer methods.

Dynamic proteome changes of Shigella flexneri 2a during transition from exponential growth to stationary phase / 基因组蛋白质组与生物信息学报·英文版

Shigella flexneri is an infectious pathogen that causes dysentery to human, which remains a serious threat to public health, particularly in developing countries. In this study, the global protein expression patterns of S. flexneri during transition from exponential growth to stationary phase in vitro were analyzed by using 2-D PAGE combined with MALDI-TOF MS. In a time-course experiment with five time points, the relative abundance of 49 protein spots varied significantly. Interestingly, a putative outer membrane protein YciD (OmpW) was almost not detected in the exponential growth phase but became one of the most abundant proteins in the whole stationary-phase proteome. Some proteins regulated by the global regulator FNR were also significantly induced (such as AnsB, AspA, FrdAB, and KatG) or repressed (such as AceEF, OmpX, SodA, and SucAB) during the growth phase transition. These proteins may be the key effectors of the bacterial cell cycle or play important roles in the cellular maintenance and stress responses. Our expression profile data provide valuable information for the study of bacterial physiology and form the basis for future proteomic analyses of this pathogen.

Construction, expression and preliminary pharmacokinetic analysis of IL-1ra mutants / 生物工程学报

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.

Expression, purification and activity analysis of anti-human transferrin receptor scFv / 生物工程学报

Human transferrin receptor (TfR) was isolated from homogenates of placental tissues by affinity chromatography on transferrin-Sepharose, and then used to screen human scFv against it from a fully-synthesized phage scFv library. After verifying the specificity, gene fragment of one of the selected scFv was inserted into the plasmid pET22b(+) and transformed into E. coli BL21(DE3) . Expression of scFv in transformant was induced with 0.5mmol/L IPTG. ELISA assay on HeLa cells showed that scFv protein could recognize and bind to TfR on the surface of HeLa cells. The scFv was purified by one-step affinity chromatography with Ni+ -NTA agarose, and injected into Kunming mouse via tail veins. This scFv was detected in brain tissues 1h later by capillary depletion method, which indicates that scFv protein can permeate through the blood brain barrier by mediation of the TfR receptor. Our works lay the foundation for the treatment of tumors and central nervous system diseases.

Design and implementation of DB sequence optimization software / 生物工程学报

TIR (Translation Initiation Region) efficiency is very important in prokaryotic expression. The TIR's efficiency is highly dependent on SD (Shine-Dalgarno) sequence, distance between SD sequence and start codon, DB (Downstream Box) sequence, TIR's second structure, codon adaptation and so on. In this paper, we designed and implemented the software to optimize DB sequence and 5' rare codons. It generated some optimization sequences by analyzing the target sequence and comparing it with 16S RNA. And the optimization sequences is sorting by number of base pairing, location of base pairing and codon adaptation. We drew up the algorithm and the core of code in this paper.

Effects of hydrodynamic on aggregates formation, growth and metabolism of HEK293 cells in suspension culture / 生物工程学报

By using the size distribution of cell aggregates, viable cell density, cell viability, specific consumption rate of glucose (q(glc)), specific production rate of lactate (q(lac)) and lactate transform rate (Y(lac/glc)) as the evaluation indexes, the effects of hydrodynamic on aggregates formation, growth and metabolism of HEK293 cells in suspension culture were examined in 250mL spinner-flasks by setting the agitation rates at 25, 50, 75 and 100r/min, respectively. It was found that agitation plays an important role in HEK293 cell aggregates formation and cell aggregates size distribution. After 7d cultivation in spinner-flasks operated at 50r/min and 75r/min, the average diameter of HEK293 cell aggregates was 201 microm and 175 microm, respectively, with the fraction of aggregates larger than 225 microm less than 10%. The cell viability was kept above 90% with the metabolic indexes, including q(glc), q(lac) and Y(lac/glc) kept constant. These results demonstrated that hydrodynamic derived from the proper agitation play a decisive role in controlling the formation and size distribution of HEK293 cell aggregates, and provided sufficient mass transfer to support the normal growth and metabolism of HEK293 cells in suspended aggregates.

Characteristics of HEK293 Cells Growth and Metabolism under Carrier-free Immobilization Culture Mode / 中国生物工程杂志

By using the cell density, cell viability, size distribution of cell aggregates,specific consumption rate of glucose (q_ glc ), specific production rate of lactate (q_ lac ), lactate transform rate (Y_ lac/glc ) and amino acids utilization as the evaluation indexes, the growth and metabolism of HEK293 cells under carrier-free immobilization culture mode were examined and compared with those of HEK293 cells cultured in static tissue flasks. It was found that HEK293 cells grown as suspended cell aggregates in spinner flasks maintained the basic growth and metabolism characteristics of HEK293 cells in stationary anchored culture, and HEK293 cells under carrier-free immobilization culture mode as suspended aggregates in stirred bioreactor facilitate perfusion performance and increase unit productivity. Cultivation of HEK293 cells in carrier-free immobilization culture mode has potential for further improving mammalian cells culture technique.

Artificial transcription factors as tools for gene expression manipulation / 生物工程学报

In this new era of the genome, the complete sequences of various organisms (from the simplest to the most complex such as human) are now available, which provides new opportunities to study biology and to develop therapeutic strategies. But the paucity of research tools that manipulate specific genes in vivo represents a major limitation of functional genomic studies. In nature, the expression of genes is regulated at the transcriptional level primarily by proteins that bind to nucleic acids. Many of these proteins, which are termed transcription factors, are typically consist of two essential yet separable modules: DNA-binding domain (DBD) and effector domain (ED). Attempts to control the gene expression by artificial transcription factors are based on the application of this rule. Among the many naturally occurring DNA-binding domains, the Cys2-His2 zinc-finger domain has demonstrated the greatest potential for the design of novel sequence-specific DNA-binding proteins. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. Ideally, these artificial DNA binding proteins could be designed to specifically target and regulate one single gene within a genome as complex as that found in human. Such proteins would be powerful tools in basic and applied research.

RNase III-prepared short interfering RNAs induce degradation of SARS-coronavirus mRNAs in human cells / 生物工程学报

SARS-associated coronavirus has been identified for the cause of Severe Acute Respiratory Syndrome, for which there is no efficacious drugs or vaccines. RNA interference (RNAi) is a process in cell to degradation specific target mRNA by double-stranded RNA. In mammalian cells, RNAi can be triggered by short interfering RNA (siRNA). RNA interference of virus-specific genes has emerged as a potential antiviral mechanism. This work evaluated if RNase III-prepared short interfering RNAs can induce specific degradation of SARS-coronavirus mRNAs in human cells. Three of SARS genes, RNA dependent RNA polymerase (RdRp), spike and nucleocapsid, were amplified with T7 promoter-flanked primers. Long length double-stranded RNA of these genes were transcribed in vitro and then were cleaved to <30bp length short interfering RNA with E. coli RNase III. These siRNAs were termed esiRNA-R, esiRNA-S and esiRNA-N respectively. RdRp, spike and nucleocapsid DNA fragments were inserted into the plasmid pGL3-Control, obtained plasmids pGL-R, pGL-S and pGL-N can express hybrid mRNAs luciferase-RdRp, spike and -nucleocapsid in cells. Above plasmids and esiRNAs were co-transfected to HEK293F cells with reference plasmid pRL-TK. Firefly luciferase and Renilla luciferase activity were measured. Hybrid mRNAs' abundance was measured using reverse transcription real-time PCR. Firefly luciferase expression of pGL-R was reduced to 13% by esiRNA-R. Expression of pGLS was reduced to 11% by esiRNA-S. Expression of pGL-N was reduced to 40% by esiRNA-N. Control esiRNAs didn't affect luciferase expression; Hybrid mRNAs' abundance was dramatically reduced by corresponding esiRNAs. RNase III-prepared short interfering RNAs induce robust and specific degradation of SARS-coronavirus mRNAs in HEK293F cells. These siRNAs could be used to inhibit SARS-coronavirus in future research.

Soluble expression and target study to brain of anti-TfR ScFv / 生物工程学报

The single-chain antibody gene (ox26-scFv) to transferrin receptor (TfR) was synthesized and amplified by three-step PCR. After sequencing, the gene was cloned into prokaryotic expression vector pTIG-Trx which carried thioredoxin (Trx) gene and a C-terminal His.tag. The Ox26-scFv proteins achieved 31% yields of total bacteria proteins at 20 degrees C, after 0.02mM IPTG induction using the strain E. coli BL21 (DE3). The soluble scFv proteins in cytoplasm suspension were about 35% and the inclusion bodies were about 65%. The soluble products were purified by immobilized metal chelation affinity chromatography (Ni-NTA), a single band with molecular weight 29 kD appeared on SDS-PAGE gel. Rat GH3 cell immunocytochemistry staining showed that Ox26-scFv protein could recognize and bind to transferrin receptor. Injected SD rats with Ox26-scFv proteins by tail veins, the antibodies were detected from brain tissues specially on the brain capillaries 4 h later which indicate that Ox26-scFv proteins have a good target function to brain capillaries and can permeate the blood-brain barrier mediated by the transferrin receptors.

The ht-PAm cDNA knock-in the goat beta-casein gene locus / 生物工程学报

The production of recombinant protein is one of the major successes of biotechnology, animal cells are required to synthesize proteins with the appropriate post-translational modifications. Transgenic animal mammary gland bioreactor are being used for this purpose. Gene targeting is a more powerful method to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cells provides an wonderful means of cell-mediated transgensis. Here we describe efficient and reproducible gene targeting in goat fetal fibroblasts to place the human tissue plasminogen activator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goat by nuclear transfer. To construct the gene targeting vector pGBC4tPA, the milk goat beta-casein genomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kb fragment including goat beta-casein gene 5' flanking sequence, and the right arm was 2.4 kb fragement including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned in the goat beta-casein gene exon 2, and the endogenous start condon was replaced by that of ht-PAm. The bacterial neomycin (neo) gene as positive selection marker gene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negative selection marker gene, was just outside the right arm. The validity of the positive-negative selection vector (PNS), was tested, and targeting homologous recombination (HR) were elevated to 5-fold with the negative selection marker using the drug GANC. The DNA fragment in which two LoxP sequence was delected effectively using Cre recombinase in vitro. Goat fetal fibroblasts were thawed and cultured to subconfluence before transfection, about 10(7) fibroblasts were electoporated at 240V, 600 microF in 0.8 mL PBS buffer containing linear pGBC4tPA. transfected cells were cultured in collagen-coated 96-wellplate for 24h without selection, then added the drug G418 (600 microg/mL) and GANC (2 micromol/L). After 12 days of selection, well separated G418r/GANCr clones were isolated and expanded in 24-wellplate. 244 clones were selected, and only 90 clones could grow and be tested by PCR screening for targeting. The primary result demonstrated that 31 targeting cell clones with homologous recombination events were obtained, and 2 cell clones was verified by DNA sequence analysis on the homologous recombination region.

Synthesis and function analysis of a new thrombopoietin (TPO) mimic peptide / 中国实验血液学杂志

In order to find a new TPO-mimic peptide with similar activity to TPO while reducing the side effects, a TPO-mimic peptide (P1) screened from a random peptide library was restructured. The new structure of the TPO mimic peptide (P2) was synthesized. After coupling P2 with Dextran 10 and performing intermolecular oxidation, dextran-coupled and dimerized form of P2 were obtained, naming D-P2 and (P2)(2) respectively. The activities of the peptides in vitro were measured with MTT method. The results showed that the EC50 of P2 was 20 nmol/L, 700 times higher than P1. The EC50 of D-P2 and (P2)(2) were 0.35 nmol/L and 0.14 nmol/L, respectively. After administrating to the mouse, the peptides increased the number of platelets in the blood circulation obviously without influence on other blood cells. In conclusion, the TPO-mimic peptides have prospects in treating diseases related with thrombocytopenia.

Expression, purification and serological reactivity of a chimeric antigen of GRA6 with P30 from Toxoplasma gondii / 生物工程学报

Major surface protein (p30) and Dense Granule Antigen GRA6 of Toxoplasma gondii have good antigenicity, and could be used for detection of IgM against Toxoplasma gondii. GRA6 may complement P30 to reach more high sensitivity for detection of antibodies to Toxoplasma gondii, so, we try to express the chimeric protein of GRA6 and P30 by genetic engineering, identify its antignenicity and use for developing diagnosis reagent. Antigenic domains of p30 and GRA6 of Toxoplasma gondii were screened by analyzing their sequences using the software ANTHEWIN. Two DNA fragments encoding respectively antigenic domains of p30 and GRA6 were cloned, they were inserted into the same expression vector pET28a( + ) and expressed as a chimeric protein in Escherichia coli. BL21(DE3), the expressed chimeric protein of p30 with GRA6 in a form of inclusion body was about 25% of total proteins of E. coli. BL21(DE3). The inclusion body was washed once with 0.5% Triton X-100 and dissolved with 0.5% SKL, after renaturation by gradient dialysis, the recombinant protein was purified by DEAE-Sepharose FF cation column and then detected with 12% SDS-PAGE, it exists mainly in the eluted peak with 300 mmol/L NaCl and has high purity. By using enzyme-linked immunosorbent assay (ELISA), the recombinant protein was examined for reactivity with immunoglobulin M (IgM) antibodies in 6 sera from patients infected with Toxoplasma gondii ., it was reactive with all the 6 sera but not with sera from normal people, these results showed that the recombinant chimeric antigen has good antigenicity and specificity and could be used for detection of IgM against Toxoplasma gondii. The expressed chimeric protein could be used for epidemic investigation of Toxoplasma gondii, blood donor screening, especially for detection of pregnant women, and is of great significance in prevention of Toxoplasma gondii infection.

Construction of a SV40 promoter specific artificial transcription factor / 生物工程学报

Transcriptions are regulated by transcription factors. Natural transcription factors usually consist of at least two functional domains: a DNA-binding domain and an effector domain. According to this, novel artificial transcription factors are designed to up or down regulate transcription and expression of a target gene. The Cys2-His2 zinc finger domain is a DNA-binding module that has been widely used as the DNA-binding domain in artificial transcription factors. Each zinc finger domain, which comprises about 30 amino acids that adopt a compact structure by chelating a zinc ion, typically functions by binding 3 base pairs of DNA sequence. Several zinc fingers linked together would bind proportionally longer DNA sequences. According to the "bipartite complementary" library strategy, a pair of zinc finger phage display libraries were constructed. After construction of the libraries, a 9bp sequence (5'-GCAGAGGCC-3') on the promoter of SV40 was chosen as a target for next step. After parallel selection, PCR amplification, desired fragments recovery, re-ligation, and additional rounds of selection, phage enzyme-linked ELISA experiments were performed to identify specific binding clones displaying the zinc fingers with predetermined sequence-specificity to our target sequence. Then two clones with strong ELISA signals were chosen to be tested for binding both to its full target site (5'-GCAGAGGCC-3') and to sites containing single transition mutations. The binding specificity of one of the two clones (clone 3) was shown to be fairly good. The three-finger DNA-binding domain targeted to SV40 promoter, that is, zinc finger sequences on clone 3, was fused to KOX1 suppression domain KRAB and cloned into pcDNA3.1 (+) (which expression product was artificial transcription factor). The zinc fingers (which expression product was the DNA-binding domain of artificial transcription factor) and KRAB domain only (which expression product was effector domain of artificial transcription factor) were also cloned separately into the same expression vector. All constructs contained an N-terminal nuclear localization signal. Every of the vectors (including pcDNA3.1 (+) without inserting sequences) were cotransfected with pGL3-Control and pRL-TK and the activity of luciferase was used to indicate the function of product from transfected expression vectors. Our artificial transcription factor was proved to repress the expression of reporter gene efficiently,while with only DNA-binding domain or effector domain the repression was not remarkable. By adding different effector domains and changing the DNA-binding domain, artificial transcription factor would have a wide range of potential applications.