[Analysis of Low C/N Wastewater Treatment and Structure by the CEM-UF Combined Membrane-Nitrification/Denitrification System].

In this study, a CEM-UF composite membrane with ammonia nitrogen enrichment and separation characteristics was combined with nitrification/denitrification to treat low C/N wastewater. The denitrification characteristics of low C/N wastewater at different flow ratios were investigated, and the structural characteristics of functional microbial communities in nitrifying and denitrifying activated sludge were analyzed by 16Sr DNA high-throughput sequencing. The results showed that influent TN was 60 mg·L-1, COD/TN was 2.65, the nitrification effect of each flow rate was good, and the average ammonia nitrogen removal rate was 98.7%. When the flow ratio increased from 1:2 to 1:6, the m(COD)/m(NO3--N) of denitrification was increased, and the removal of average nitrate nitrogen reached its highest level at 1:6, which was 86.28%, and the removal of total nitrogen increased from 22.56% to 46.8%. An analysis of Illumina sequencing showed that nitrogen fixing bacteria Proteobacteria accounted for 30.9%, and the important nitrite oxidizing bacteria, Nitrospirae, accounted for 3.06%. At the genus level, Nitrosomonas and Nitrosospira, belonging to the ammonia oxidizing bacteria (AOB) category and Nitrospira and Nitrobacter, belonging to the nitrite oxidizing bacteria (NOB) category were detected. The ratio of AOB and NOB bacteria was high, which is consistent with good nitrification in the nitrification reactor. The dominant bacteria in denitrification sludge were Proteobacteria (53.13%), followed by Bacteroidetes (10.93%). A variety of bacteria related to denitrification were detected at the genus level, such as Dechloromonas, Thauera, Castellaniella, Alicycliphilus, Azospira, Comamonas, Caldilinea, and Saccharibacteria. The proportion of denitrifying bacteria was 25.91% as denitrifying bacteria microbial species were rich in the denitrifying sludge, giving a good denitrification effect.

Polymorphisms in genes of the de novo lipogenesis pathway and overall survival of hepatocellular carcinoma patients undergoing transarterial chemoembolization.

Aberrant expression of genes in de novo lipogenesis (DNL) pathway were associated with various cancers, including hepatocellular carcinoma (HCC). Single nucleotide polymorphisms (SNPs) of DNL genes have been reported to be associated with prognosis of some malignancies. However, the effects of SNPs in DNL genes on overall survival of HCC patients receiving transarterial chemoembolization (TACE) treatment are still unknown. In present study, nine SNPs in three genes (ACLY, ACACA and FASN) in DNL pathway were genotyped using the Sequenom iPLEX genotyping system in a hospital-based cohort with 419 HCC patients treated with TACE, and their associations with HCC overall survival were evaluated by Cox proportional hazard regression analysis under three genetic models (additive, dominant and recessive). Although we did not find any significant results in total analysis (all p>0.05), our stratified data showed that SNP rs9912300 in ACLY gene was significantly associated with overall survival of HCC patients with lower AFP level and SNP rs11871275 in ACACA gene was significantly associated with overall survival of HCC patients with higher AFP level. We further identified the significant interactions between AFP level and SNP rs9912300 or rs11871275 in the joint analysis. Conclusively, our data suggest that genetic variations in genes of DNL pathway may be a potential biomarker for predicting clinical outcome of HCC patients treated with TACE.

Fatty acid synthesis pathway genetic variants and clinical outcome of non-small cell lung cancer patients after surgery.

Over-expression of de novo lipogenesis (DNL) genes is associated with the prognosis of various types of cancers. However, the effects of single nucleotide polymorphisms (SNPs) in these genes on recurrence and survival of non-small cell lung cancer (NSCLC) patients after surgery are still unknown. In this study, a total of 500 NSCLC patients who underwent surgery treatment were included. Eight SNPs in 3 genes (ACACA, FASN and ACLY) of the DNL pathway were examined using the Sequenom iPLEX genotyping system. Multivariate Cox proportional hazards regression and Kaplan-Meier curves were used to analyze the association of SNPs with patient survival and tumour recurrence. We found that two SNPs in the FASN gene were significantly associated with the recurrence of NSCLC. SNP rs4246444 had a significant association with lung cancer recurrence under additive model (hazard ratio [HR], 0.82; 95% confidence interval [95%CI], 0.67-1.00; p=0.05). Under the dominant model, rs4485435 exhibited a significant association with recurrence (HR, 0.75; 95%CI, 0.56-1.01; p=0.05). Additionally, SNP rs9912300 in ACLY gene was significantly associated with overall survival in lung cancer patients (HR, 1.41; 95%CI, 1.02-1.94, p=0.04) under the dominant model. Further cumulative effect analysis showed moderate dose-dependent effects of unfavorable SNPs on both survival and recurrence. Our data suggest that the SNPs in DNL genes may serve as independent prognostic markers for NSCLC patients after surgery.

Leukocyte telomere length predicts overall survival in hepatocellular carcinoma treated with transarterial chemoembolization.

Previous studies have reported that telomere length in peripheral blood leukocytes can predict the clinical outcome of several cancers. However, whether leukocyte telomere length is associated with the prognosis of hepatocellular carcinoma (HCC) remains to be determined. In this study, relative telomere length (RTL) in peripheral blood leukocytes was measured using a real-time PCR-based method for 269 HCC patients treated with transarterial chemoembolization (TACE) from two independent hospitals. The association between RTL and the overall survival (OS) of HCC was analyzed. The immunological function of the HCC patients with different leukocyte RTLs was evaluated. Multivariate analyses indicated that long leukocyte RTL was significantly associated with poor OS of HCC patients, with a hazard ratio of 2.04 (95% confidence interval, 1.46-2.86; P < 0.001). Kaplan-Meier analyses showed a significant difference of median survival time between patients with long and short RTL (log rank P < 0.001). Fluorescence-activated cell sorting analyses showed that the long RTL group had a significantly increased percentage of CD4(+)CD25(+)FOXP3(+) Treg in CD4(+) T cells compared with short RTL group (P = 0.002). In conclusion, our results suggest that leukocyte RTL may serve as an independent prognostic marker for HCC patients treated with TACE.

Characterization of basigin isoforms and the inhibitory function of basigin-3 in human hepatocellular carcinoma proliferation and invasion.

Basigin, which has four isoforms, plays an important role in invasion of hepatocellular carcinoma (HCC). Detailed transcriptional regulation and functions of the basigin isoforms have not been reported except in the case of the predominant isoform basigin-2, which act as inducer of matrix metalloproteinases (MMPs). Here we determined that basigin-2, basigin-3, and basigin-4 were the most abundant transcript variants in human cell lines. GeneRacer PCR and luciferase reporter assays showed that basigin-3 and basigin-4 were initiated from an alternative promoter. Basigin-3 and basigin-4 were widely expressed in various normal human tissues at the mRNA level and were upregulated in HCC tissues compared to in normal tissues. Western blotting and confocal imaging showed that glycosylated basigin-3 and basigin-4 were expressed and localized to the plasma membrane. However, in cultured cell lines, only native basigin-3, and not basigin-4, was detected at protein level. Overexpression of basigin-3 inhibited HCC cell proliferation, MMP induction, and cell invasion in vitro and in vivo. Bimolecular fluorescence complementation assays and nuclear magnetic resonance (NMR) analysis indicated that basigin-3 interacted with basigin-2 to form hetero-oligomers. In conclusion, we systematically investigated the alternative splicing of basigin and found that basigin-3 could inhibit HCC proliferation and invasion, probably through interaction with basigin-2 as an endogenous inhibitor via hetero-oligomerization.

Longer leukocyte telomere length predicts increased risk of hepatitis B virus-related hepatocellular carcinoma: a case-control analysis.

BACKGROUND: Convincing evidence has indicated that an alteration in telomere length is involved in tumorigenesis. In epidemiologic studies, a strong correlation also has been observed consistently between relative telomere length (RTL) in peripheral blood leukocytes (PBLs) and susceptibility of many cancers. However, whether leukocyte RTL can be used as a predictor of risk for hepatocellular carcinoma (HCC) remains to be determined. METHODS: The RTL in PBLs was determined by measuring the telomere repeat copy number to single-copy gene number ratio in each sample compared with a reference DNA sample using a polymerase chain reaction-based method in this case-control study. The study participants included 240 patients with HCC (cases), a group of 240 healthy individuals (controls), and 120 noncancer controls with chronic liver disease (CLD). RESULTS: HCC cases exhibited a significantly longer RTL (median, 0.57; range, 0.21-3.3) than CLD controls (median, 0.46; range, 0.15-1.99; P < .001) and healthy controls (median, 0.39; range, 0.13-2.69; P < .001). Compared with individuals who had short RTL, individuals who had long RTL had a significantly increased risk of HCC when either healthy controls (adjusted odds ratio [OR], 7.28; 95% confidence interval, 4.46-11.88) or CLD controls (adjusted OR, 2.86; 95% confidence interval, 1.74-4.70) were used as the reference group. A significant dose-response relation was observed between HCC risk and long RTL (P(trend) < .001 for both control groups). In addition, there was a significantly positive RTL correlation between PBLs and normal liver tissues (r = 0.78; P < .001) or cirrhotic liver tissues (r = 0.67; P = .001). Furthermore, a significant joint effect on the risk of HCC was noted between RTL and smoking status or alcohol use. CONCLUSIONS: The current study produced the first epidemiologic evidence linking long RTL in PBLs to an increased risk of HCC. The authors concluded that these findings warrant further investigation in other populations.

Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma.

CD147 is a transmembrane glycoprotein overexpressed in human hepatocellular carcinoma (HCC) which could promote HCC progression and metastasis. Promoter methylation is one of the most important processes in gene regulation. In this study, we aim to investigate CD147 promoter methylation status and the correlation with clinicopathological features and prognosis in HCC. CD147 promoter methylation statuses and expression levels in normal and HCC cell lines and 54 paired HCC and adjacent non-tumour (ANT) tissues were, respectively, examined by bisulphite genomic sequencing, methylation-specific PCR, real-time RT-PCR, Western blot and immunohistochemistry. The correlations of promoter methylation statuses with CD147 expression level and the clinicopathological features were statistically analysed in HCC patients. Significantly higher expression of CD147 and significantly lower promoter methylation level were observed in HCC cell lines compared to normal cell lines and tissues control. In vivo and in vitro analysis indicated that demethylation with 5-Aza-2'-deoxycytidine led to increased CD147 expression through enhancing Sp1 binding affinity, and methylation with methyltransferase reduced CD147 transcriptional activity through interfering Sp1 binding. CD147 promoter methylation level in HCC tissues (22.22%) was lower than that in ANT tissues (46.30%; P < 0.05). Within HCC tissues, a significant inverse correlation was observed between CD147 expression and methylation level (r=-0.615). Moreover, HCC patients with unmethylated CD147 promoter had a significantly higher recurrence rate (88.1%versus 58.3%; P < 0.05) and death rate (83.3%versus 50.0%; P < 0.05) than patients with methylated CD147 promoter. In conclusions, promoter hypomethylation up-regulates CD147 expression primarily through increasing Sp1 binding and associates with poor prognosis in HCC patients.

Transcription factor Sp1 regulates expression of cancer-associated molecule CD147 in human lung cancer.

CD147 is a novel cancer-associated biomarker that plays an important role in the invasion and metastasis of human lung cancer. In spite of its many known functions, little is known about CD147 transcriptional regulation. In this study, we explored the regulation of CD147 in human lung cancer tissues. Over 60% of the human lung cancer tissues expressed differential high levels of CD147. We then cloned the 5'-flanking region of the human CD147 gene and identified a critical promoter region at -108 to -42 which contained one binding site for Sp1, which was essential in up-regulating CD147 promoter activity. These results were proven by blocking Sp1 using RNAi or mithramycin A treatment and up-regulating Sp1 using transfection with eukaryotic expression vector. Consistent with the CD147 transcription activation, a high level of Sp1 expression was detected in lung cancer cell lines overexpressing CD147. Chromatin immunoprecipitation assay showed that much more Sp1 could bind to the CD147 promoter in 95-D with CD147 high expression than in SK-MES-1 with CD147 low expression. There was a significant positive correlation between CD147 expression and Sp1 expression level detected by immunohistochemistry (r = 0.831). Collectively, our results suggest that Sp1 is essential for regulating the CD147 gene expression in human lung cancer.

HAb18G/CD147 functions in invasion and metastasis of hepatocellular carcinoma.

CD147 molecule is reported to be correlated with the malignancy of some cancers; however, it remains unclear whether it is involved in the progression of hepatocellular carcinoma (HCC). Here, we investigated the function of HAb18G/CD147, a member of CD147 family, and its antibodies, HAb18 and LICARTIN, in HCC invasion and metastasis. We observed that HAb18G/CD147 gene silence in HCC cells significantly decreased the secretion of matrix metalloproteinase (MMP) and the invasive potential of HCC cells (P < 0.001). MMP silence in HCC cells also significantly suppressed the invasion of the cells when cocultured with fibroblasts; however, its inhibitory effect was significantly weaker than that of both HAb18G/CD147 silence in HCC cells and that of MMP silence in fibroblasts (P < 0.001). Blocking theHAb18G/CD147 molecule on HCC cells with HAb18 monoclonal antibody resulted in a similar suppressive effect on MMP secretion and cell invasion, but with no significant effects on the cell growth. (131)I-labeled HAb18 F(ab')(2) (LICARTIN), however, significantly inhibited the in vitro growth of HCC cells (P < 0.001). In an orthotopic model of HCC in nude mice, HAb18 and LICARTIN treatment effectively reduced the tumor growth and metastasis as well as the expression of three major factors in the HCC microenviroment (MMPs, vascular endothelial growth factor, and fibroblast surface protein) in the paracancer tissues. Overall, these results suggest that HAb18G/CD147 plays an important role in HCC invasion and metastasis mainly via modulating fibroblasts, as well as HCC cells themselves to disrupt the HCC microenviroment. LICARTIN can be used as a drug targeting to HAb18G/CD147 in antimetastasis and recurrence therapy of HCC.

Targeting radioimmunotherapy of hepatocellular carcinoma with iodine (131I) metuximab injection: clinical phase I/II trials.

PURPOSE: HAb18G/CD147 is a hepatocellular carcinoma (HCC)-associated antigen. We developed iodine (131I) metuximab injection (Licartin), a novel 131I-labeled HAb18G/CD147-specific monoclonal antibody Fab'2 fragment, and evaluated its safety, pharmacokinetics, and clinical efficacy on HCC in Phase I/II trials. METHODS AND MATERIALS: In a Phase I trial, 28 patients were randomly assigned to receive the injection in 9.25-, 18.5-, 27.75-, or 37-MBq/kg doses by hepatic artery infusion. In a multicenter Phase II trial, 106 patients received the injection (27.75 MBq/kg) on Day 1 of a 28-day cycle. Response rate and survival rate were the endpoints. RESULTS: No life-threatening toxic effects were found. The safe dosage was 27.75 MBq/kg. The blood clearance fitted a biphasic model, and its half-life was 90.56-63.93 h. In the Phase II trial, the injection was found to be targeted and concentrated to tumor tissues. Of the 73 patients completing two cycles, 6 (8.22%) had a partial response, 14 (19.18%) minor response, and 43 (58.90%) stable disease. The 21-month survival rate was 44.54%. The survival rate of progression-free patients was significantly higher than that of patients with progressive disease after either one or two cycles (p < 0.0001 or p = 0.0019). CONCLUSION: Iodine (131I) metuximab injection is safe and active for HCC patients.

Isolating human antibody against human hepatocellular carcinoma by guided-selection.

OBJECTIVE: With the pComb3X-displaying Fab antibody libraries, to achieve the humanization of murine HAb18 against HCC by guided selection. METHODS: With the optimized primers, the human Fd and C(L) repertoire genes were amplified by RT-PCR from PBMC of HCC patients. The Fd repertoire genes were paired with murine HAb18 C(L) gene to construct pComb3X-displaying hybrid Fab library. The recombinant HAb18GE was used as antigens to select the target antibodies and got the Fd fragments. Then the human C(L) genes were paired with the selected human Fds to construct human Fab library. After the panning, the complete human Fab antibodies were got and analyzed. RESULTS: With the murine HAb18 C(L) gene as template, the heavy chain Fd shuffling was achieved by panning the hybrid Fab library. Then with the selected Fds as template, the human Fabs were obtained through the light chain shuffling. Two of the resulting human Fabs (HuFab2 and HuFab11), with same Fd and different light chains, bound to HAb18G/CD147 specifically. The competitive ELISA, Western blotting, FCM, fluorescent cell staining and so on demonstrated that the human Fabs resembled its parental murine Fab in that they both perhaps recognized the same epitope. K(D) indicated (HuFab2=210 nm and HuFab11=280 nm) the selected Fabs had available affinity. CONCLUSION: Through guided-selection, we got the available human Fab antibodies for the subsequent research. These results suggest that guided selection is a promising strategy in murine mAb humanization.

[High expression of hepatoma associated antigen HAb18G in prokaryotic cells and study on its function in vitro].

AIM: To construct prokaryotic expression vector of HAb18G, and express high level of this fusion protein in E. coli and to identify its function and immunogenicity. METHODS: The HAb18G full length cDNA from pBluescript/HAb18G was obtained by PCR and cloned into prokaryotic expression vector pRSET-C and then transformed into E. coli BL21(DE3) to induce its expression. Expressed products were analyzed by SDS-PAGE and laser thin layer scan. The purified HAb18G protein was identified by gelatin enzymogram and ELISA. RESULTS: Endonuclease digestion and DNA sequencing proved that HAb18G cDNA was cloned correctly into the expression vector. Result of SDS-PAGE showed that the relative molecular mass of the expressed product HAb18G fusion protein was 34,600, which was in accordance with predicted relative molecular mass value. Laser thin layer scan showed that the expressed product accounted for 33% of the total bacteria protein. Result of enzymogram was negative whereas the result of ELISA was positive. CONCLUSION: It was testified that the protein HAb18G has immunogenicity but no bioactivity. The high level prokaryotic expression of HAb18G lay the foundation for manufacturing the HAb18G protein in great quantities and proceeding to its relative research.

[Secretory expression of chimeric Fab antibody HAb18 against human hepatocellular carcinoma in Pichia pastoris].

AIM: To express secretively chimeric Fab antibody HAb18 (cFab) against human hepatocellular carcinoma in Pichia pastoris. METHODS: Genes encoding CL chain and Fd fragment of cFab antibody HAb18 were subcloned into vectors pPIC9K and pPICZalphaA, respectively. After confirmed by DNA sequence analysis, the recombinant plasmids pPIC9K/CL and pPICZalphaA/Fd were transformed into the genome of Pichia pastoris GS115. Mut(+) multiple insert transformants were screened by G418 and Zeocin and then induced with 5 mL/L methanol to express cFab. RESULTS: 4 days after methanol induction, 26 mg/L of the cFab fragment was detected in the culture supernatant. Western blot proved that the expressed protein could specifically bind with HAb18GEF antigen. CONCLUSION: The successful expression of cFab/HAb18 in Pichia pastoris lays the foundation for large-scale production and further application of the antibody.

[Comparison of polyclonal anti-sera against extracellular domain of hepatoma associated antigen HAb18G/CD147 prepared by different immunization schemes].

AIM: To compare characteristics of polyclonal anti-sera against extracellular domain of hepatoma-associated antigen HAb18G/CD147(HAb18GEF) generated by different immunization schemes. METHODS: BALB/c mice were immunized with GST-HAb18GEF fusion protein expressed in E.coli (routine immunization method), recombinant eukaryotic expression plasmid pcDNA3/HAb18G (intramuscular injection) and pcDNA3/HAb18G plasmid followed by human hepatoma cells booster (DNA-cell booster), respectively. The titers and Ig subclasses of polyclonal anti-sera against denatured and natural HAb18GEF were detected by indirect ELISA and cell ELISA, respectively. The specific binding of polyclonal anti-sera prepared by different immunization schemes to denatured HAb18GEF was analyzed by Western blot. The specific binding of polyclonal anti-sera produced by DNA-cell booster immunization to natural HAb18G antigen on hepatoma cells was detected by immunofluorescence staining. RESULTS: GST-HAb18GEF immunization could induce polycolonal antibody IgG1 with higher titer mainly against denatured or linear epitopes on HAb18GEF. Antibody induced by pcDNA3/HAb18G intramuscular immunization was IgG2a with lower titer against natural epitopes on HAb18GEF. DNA-cell booster immunization could induce generation of polycolonal antibody IgG2a and IgG1 of moderate titers against the native epitopes on HAb18G. CONCLUSION: The polycolonal sera with different titers against different epitopes on HAb18GEF can be induced by different immunization schemes.

Prokaryotic expression and renaturation of engineering chimeric Fab antibody against human hepatoma.

AIM: To express chimeric Fd (cFd) and chimeric light chain (cL) in E.coli respectively and refold them into chimeric Fab (cFab) antibody. METHODS: cFd and cL genes were respectively inserted into the prokaryotic expression vector pET32a to construct recombinant vectors pET32a/cFd and pET32a/cL. Then, the competent E.coli cells were transformed by the recombinant vectors and induced by IPTG. Moreover, a large quantity of cFd and cL expression products were prepared and mixed with equal molar to refold into cFab by gradient dialysis. The refolded products were identified and analyzed by sodium SDS-PAGE, Western blotting, ELISA and HPLC. RESULTS: High efficient prokaryotic expressions of both cFd and cL in the form of non-fusion protein were obtained with the expression levels of 28.3% and 32.3% of total bacteria proteins, respectively. Their relative molecular masses were all 24 ku or so, and both of them mainly existed in the form of inclusion bodies. In addition, cFd and cL were successfully refolded into cFab by gradient dialysis, with about 59.45% of recovery when the starting total protein concentration was 100 microg/mL. The renatured cFab could specifically bind to related antigen with high affinity. CONCLUSION: The cFab antibody against human hepatoma was highly and efficiently expressed and refolded, which laid a solid foundation for studying its application in the treatment of hepatoma.

[Preparation of human Fab antibodies against HAb18G by guided selection and chain shuffling technique].

AIM: To prepare humanized Fab antibody by guided selection and chain shuffling technique. METHODS: Human Fd and C(L) repertoire genes were amplified by RT-PCR from PBMCs of patients with hepatocellular carcinoma. Human-murine chimeric C(L) genes were paired with the human Fd repertoire genes to construct phage antibody library. After four rounds of panning against HAb18GE, chimeric antibodies containing humanized Fd gene and binding to HAb18GE were obtained. Then the selected Fd genes were paired with the human C(L) repertoire genes to construct a humanized Fab antibody gene library. After four rounds of panning, completely humanized Fab antibodies were obtained. RESULTS: After six rounds of panning, 7 chimeric Fab genes were obtained from the Fab gene library with 2 x 10 (7) pfu. Using the selected seven Fd genes, the humanized Fab gene library of 0.8 x 10 (7) pfu was constructed. After four rounds of panning, 2 humanized Fab genes with the strongest reactivity to HAb18G were obtained. DNA sequencing showed that Fds of the two humanized Fabs had the same DNA sequence, which belonged to IgG2, just the same as the parent antibody. And the C(L) belonged to kappa type, and V kappa 3 family. CONCLUSION: By guided-selection and chain shuffling technique, the humanized Fab antibody against HAb18G is obtained, which lays the foundation for further research.

[Non-fused expression of HAb18GEF by reducing stability of translational initiation region in mRNA].

To express the extracellular fragement of hepatoma associated antigen HAbl8G(HAb18GEF) in E. coli efficiently in a non-fusing way, the cDNA of HAb18GEF gene was inserted into prokaryotic expression vector pET21a + . The secondary structure and codon adaptation of translational initiation region (TIR, from-30 to + 39) in mRNA of recombinant vector HAb18GEF/ pET21a + was predicted simultaneously by computer-aided design. Stable Stem-Loop structures and many low-usage codons were detected in mRNA-TIR of non-optimized recombinant HAb18GEF/pET21a + vector. The stability of mRNA-TIR in recombinant HAb18GEF/pET21a + vector was reduced with following methods: (1) optimization of secondary structure (2) optimization of codon adaptation. These optimization were realized by non-continual site-directed mutagenesis without changing any amino acid sequence in TIR. After being checked through restriction endonuclease digestion and confirmed through nucleotide sequencing, the pre-optimized and post-optimized recombinant vectors were transformed into competent E. coli JM109-DE3. The resulted recombinant clones were selected randomly and induced by IPTG at 37 degrees C. The induced production of these recombinants was analyzed by SDS-PAGE, indirect ELISA, Western blot, and cell fractionation assay. The amount of HAb18GEF mRNA was also detected by RNA dot blot between pre-optimized recombinant and post-optimized recombinant. The results revealed that recombinant non-fused vectors HAb18GEF/pET21a + were successfully constructed and optimized in the secondary structure and codon adaptation of TIR respectively. The HAb18GEF was expressed efficiently in a non-fusing way in recombinant E. coli by secondary structure optimization or codon adaptation optimization. Whereas, no expression of HAb18GEF was detected in pre-optimized recombinants. The non-fused expression products-HAb18GEF, mainly as inclusion body in E. coli, yielded highly above 29.3%. A trait of expression HAb18GEF was also detected both in intermembrane space and in culture medium due to over-expression and cell leakage. Difference in non-fused expression level of HAb18GEF between secondary structure optimization and codon adaptation optimization was negligible. No difference in amount of transcribed mRNA of HAb18GEF between the pre-optimized and the post-optimized recombinants was detected. To sum up, it's feasible to express hepatoma associated antigen HAb18GEF in a non-fusing way by reducing the stability of TIR in mRNA.

[Construction and characterization of anti-DOTA-Y phage antibody library].

AIM: To construct a anti-dodecane-tertraacetic acid-yttrium(DOTA-Y) immune Fab phage antibody library. METHODS: BALB/c were immunized with BSA-Y-DOTA which was prepared by DOTA-conjugated BSA and chelated with Y. After determination of anti-serum, total RNA was extracted from splenic lymphocytes of immuned mice. The heavy chain Fd and light chain Kappa genes repertoires of immunoglobulin were amplified respectively by RT-PCR, and then the amplified products were cloned into the reconstructive phage vector pComb3M to construct anti-DOTA-Y Fab antibody. And then, the recombination rate, diversity and display of Fab antibody library were identified by restriction endonuclease digestion, DNA sequencing and ELISA. RESULTS: BSA-Y-DOTA was prepared successfully, and a higher titer of immune sera was achieved. The amplified gene fragments of Fd and Kappa chain by RT-PCR were correct and the length was with about 650 bp, and were inserted exactly. The sink size of Fab phage display library reached 8 x 10(7), the re-combination rate was about 90%, and it possesed great diversity. In addition, ELISA detection showed that there was Fab expression on the phage library. CONCLUSION: An immune Fab phage antibody library of DOTA-Y has been constructed successfully, which lays a solid foundation for screening specific anti-DOTA-Y antibody.

[Cloning and expression of Fab gene of mAb HAb18 against human hepatocellular carcinoma].

AIM: To clone Fab gene of mAb HAb18 against human hepatocellular carcinoma and express it in E.coli. METHODS: The cDNAs of kappa chain and Fd of mAb HAb18 were amplified by RT-PCR and cloned into prokaryotic expression vector pComb3 which was transfected into competent E.coli to express Fab by IPTG induction. The specificity of the expressed Fab was tested by ELISA and immunofluorescence staining. RESULTS: The Fab gene of mAb HAb18 was successfully amplified and expressed in E.coli. The results of competitive ELISA and immunofluorescence staining showed that the expressed Fab had specific antigen-binding activity. CONCLUSION: HAb18 Fab was prepared successfully, which lays the foundation for its further application to diagnosis and therapy of human hepatocellular carcinoma.