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Objective To systematically study the efficacy and safety of KRASG12C inhibitors in advanced solid tumors with KRASG12C-mutated. Methods Computer searches from PubMed, The Cochrane Library, Web of Science, Embase, CNKI, and CBM databases were conducted to collect clinical studies on KRASG12C inhibitors in advanced solid tumors with KRASG12C-mutated, with a search time from inception to October 12, 2022. Then, two investigators independently screened the literature, extracted information, assessed the risk of bias in included studies, and performed meta-analyses using RevMan 5.4 software. Results There were four publications included, all of which were single-arm clinical studies. The KRASG12C inhibitors that completed clinical phase Ⅰ and Ⅱ trials were sotorasib and adagrasib, with two publications each. A total of 388 and 394 patients were included in the efficacy evaluation and safety evaluation, respectively. Resultsof the Meta-analysis showed that the patients had objective response rate, overall disease control, and disease stabilization rates of 35%, 82%, and 45%, respectively. In addition, the rate of serious adverse events, general adverse events, and all adverse events in patients was 2%, 28%, and 79%, respectively. Moreover, the rate of partial remission of disease in NSCLC patients was 38%. Conclusion The KRASG12C inhibitors sotorasib and adagrasib exhibited good efficacy and high safety in advanced solid tumors.
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Optical molecular imaging is more and more widely used in the field of biomedical sciences due to its advantageous properties such as non-invasive, real-time and high resolution. As a kind of important optical molecular imaging probe,the near-infrared fluorescent(NIRF)dyes exhibit less tissue absorption and strong tissue penetration,and has been gradually applied to the early diagnosis of tumors. Researchers have developed a number of NIRF dyes with high potential for clinical application by conjugating tumor-targeting ligands,nano-modifications and multimodal NIRF imaging, and have significantly improved the specificity and sensitivity of these NIRF dyes in tumor diagnosis. In this paper we provide a review on the application of NIRF dyes in tumor diagnosis.
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The patient-derived orthotopic xenograft (PDOX) model better maintains the genetic characteristics of the primary tumor, and keep stable in histology, transcriptome, polymorphism and copy number variations. It also retains the interaction between the tumor mesenchyma, microvessels and stroma, and the tumor metastatic properties. Therefore, PDOX model can predict disease prognosis more accurately and can be used to screen appropriate individualized treatment strategies, thus, shows perfect prospect in clinical application. However, due to the differences between mouse and human microenvironment, morphological distinctions between orthotopic xenograft tumors and primary tumors still exist, and tumor metastasis can not be ensured by orthotopic xenograft. Thus, in order to construct the individualized PDOX model and to promote its clinical translation, it is necessary to analyze the histomorphology of orthotopic xenografts, to establish database of the transplantation model and share the information of the model. In this review, we will summarize the main features of PDOX models with its advantages and limitations, and looking forward to its application in the future.
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The high-fidelity prostate tumor patient-derived xenograft ( PDX) model is the basis for studies of biology and pharmacotherapy of prostate cancer. However, the development and application of prostate tumor has been hampered by a low success rate of transplanted primary tumors in mice as most prostate cancers are highly relevant to hormones. The high-fidelity PDX model of prostate cancer better maintains the histopathology and molecular heterogeneity of the original tumor. Here, we review the improved method of establishing PDX model of prostate cancer, including the testosterone supplementation, the quality of the original tumor tissue as well as the stromal niche, and the application of commonly used therapeutic drugs, and to provide a theoretical basis for clinical studies of prostate tumor. These attempts are very important for development of new agents and research on mechanisms of prostate cancer. It will further promote the individualized treatment of prostate cancer.
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Objective To establish and evaluate a patient-derived orthotopic xenograft ( PDOX ) model of pancreatic cancer. Methods Tissues of patient-derived pancreatic tumor were transplanted into nude mouse pancreas by surgery. The PDOX models were evaluated by the small animal near infrared fluorescence ( NIRF) optical imaging and PET/CT. The traceability of PDOX models was detected by STR technology, and the pathological changes were observed by H&E staining, immunohistochemistry, and serum level of CA19-9 was detected by ELISA. Results Apparent NIRF were observed to be accumulated in pancreatic site by optical imaging system. The location and size of the xenografts tumor were revealed by fluorescence intensity. The PET/CT images with 18F-FDG molecular probe confirmed the tumor's location and size. Ex vivo NIRF imaging of isolated organ further showed the tumor formation. The traceability of PDOX models was 99. 99% with human origin. H&E staining pathology and immunohistochemistry indicated the pancreatic cancer characteristics. The high serum level of ca19-9 confirmed the mice bearing tumor. Conclusions Pancreatic PDOX models are successfully established in this study, and it can be evaluated comprehensively by NIRF optical imaging and PET/CT, providing an appropriate platform for further research of pancreatic cancer.
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Multi-modal fusion molecular imaging technology integrates the advantages of a variety of molecular imaging techniques,and has become a hotspot and trend in the field of molecular imaging. Heptamethine cyanine dye is a class of novel near-infrared fluorescence(NIRF)dye with tumor targeting properties. With its unique optical properties, the dye has broad application prospects in tumor molecular imaging, targeted therapy and drug delivery system. Nano-materials containing heptamethine cyanine dye can be used for NIRF/MRI dual-modal imaging. NIRF/PET dual-modal imaging can be achieved after labeling with nuclides. Conjugated with chemotherapy drugs,targeted delivery of anti-tumor drugs can also be achieved. Complexes of multiple heptamethine cyanine dyes have been used for multi-modal imaging as a new strategy for photothermal therapy,photodynamic therapy and combined treatment of tumors.
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Objective To evaluate the therapeutic effect of chemotherapeutic drugs on pancreatic carcinoma based on patient-derived xenograft(PDX)models,and to screen an individualized treatment strategy. Methods Fresh human pancreatic carcinoma tissues were subcutaneously transplanted into nude mice to establish PDX models which could be stab-ly passaged. The traceability of PDX models was determined by STR analysis. The PDX models were treated with three dif-ferent clinical chemotherapeutic drugs oxaliplatin, gemcitabine and irinotecan, respectively, and the tumor volumes were measured at different times. The therapeutic effect of those drugs was assessed by TGD mathematical model and plasma CA19-9 test. Results The traceability of patient-derived xenograft samples was up to 99.99%. Compared with the con-trol group,the treatment with irinotecan and gemcitabine inhibited tumor growth significantly(P=0.001), and gemcit-abine had even better result. The minimum toxic effect in the mice was induced by irinotecan treatment,followed by gem-citabine treatment. Conclusions Pancreatic carcinoma PDX models are successfully established and can be stably pas-saged. Gemcitabine shows the most inhibitory effect on tumor growth based on TGD mathematical model assessment, and deserves to be recommended as the preferred drug for individual treatment of pancreatic carcinoma.
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Objective To study the tumor targeting ability and application of farnesylthiosalicylic Acid (FTS) and heptamethine carbocyanine fluorescent dye-mediated near-infrared imagine in living animals, and confirm the inhibitory effect of this compound on growth of tumor cells.Methods Human breast cancer cell line MCF-7, glioma cell line U251 and prostate cancer cell line PC3 were cultured to logarithmic growth phase, and different concentrations of FTS and FTS-IR783 were added, respectively.We observed the inhibitory effect of those two compounds on the growth of tumor cells.Under fluorescence microscopy, specific accumulation of FTS-IR783 in these tumor cells was observed.The tumor cells (1×106) were transplanted subcutaneously into nude mice.These mice were subjected to intraperitoneal injection of FTS-IR783 (10 nmol/mouse) two weeks later.In the in vivo imaging, near infrared fluorescence signal and tumor volume were measured and their correlation was analyzed.Results Compared with FTS, FTS-IR783 significantly inhibited the growth of MCF-7, U251 and PC3 cells in vitro.FTS-IR783 was specifically uptaken by these three kinds of tumor cells, showing strong near infrared fluorescence in cell agglomerates.After subcutaneous injection of FTS-IR783, the correlation between fluorescence intensity and tumor volume was 0.987, 0.998 and 0.971, respectively.Conclusions The compound of FTS conjugated with near infrared fluorescent dye IR-783 can specifically recognize tumor cells, in both in vitro and in vivo imaging.At the same time, the compound can significantly inhibit the growth of tumor cells, and may be expected to become a new potential targeted drug.
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Objective To study the application of hepatamethine cyanine near-infrared fluorescence (NIRF) dye IR-783 in the mouse models of human liver cancer exenografts, and to analyze the molecular mechanisms of the NIRF dye targeting tumor cells.Methods Luciferase-tagged HepG2 cells were inoculated subcutaneously into the nude mice.We detected the correlation of NIRF intensity and bioluminescence intensity (BIL) in the tumor region.Patient-derived xenograft (PDX) model was established in mouse by subrenal capsular implantation of clinic liver cancer specimen.After injecting the IR-783 dye, the interface between mouse kidney and the xenograft tumors was confirmed by NIRF analysis, and the tumor tissue in kidney was observed by pathology using H&E staining.The expression of CEA, AFP, HIF1α and OATP3A1 in the liver cancer tissue was detected by immunohistochemical staining.The intracellular retention of NIRF dyes was observed under fluorescence microscope after adding Mito Tracker or Lyso Tracker into cultured HepG2 cells.We added IR-783 in a co-culture system of HCCs and normal liver cells to test the specifical identification ability of IR-783 of the liver cancer cells.Results There was a good correlation between NIRF intensity and BIL intensity of the subcutaneous liver cancer xenograft region in nude mice.The margin between the mouse kidney tissue and xenograft tumors was clearly identified by IR-783.Compared with normal kidney tissue, CEA, HIF1α, OATP3A1 and AFP were highly expressed in the tumor region detected by IHC staining.The NIRF dye IR-783 was mainly accumulated in the mitochondria and lysosomes of cancer cells.GFP-tagged HepG2 cells could be recognized directly, whereas red fluorescence was not detected in normal liver cells.Conclusions IR-783 is a novel near-infrared fluorescent dye with tumor targeting and imaging properties.Its targeting ability may be related to the high expression of HIF1α and OATP3A1 in the liver cancer tissue.
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Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank .sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C 57BL/6 mice.After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile , real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice .Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas 9.After microinjection, miRNA-29b1 gene-mutated mice were obtained.The sequencing results showed that there were two types of genotype for the mutated mice , one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion.Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly .Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR /Cas9 technology.
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Objective To knockout Rag2 and IL2rg genes and construct severe combined immunodeficiency mice based on CRISPR/Cas9 technology. Method Design and synthesis of 25 bp sgRNA were made according to the Rag2 and IL2rg sequences in Genbank. After annealing, sgRNA was cloned into pX330 vector. Recombination plasmid Rag2?sgRNA, IL2rg?sgRN and Cas9 were then transcribed into RNA, these RNA were microinjected into zygotes and the zygotes were transplanted into recipient ICR mice. F0 founders were born and mutated F0 founders mated with wild type mice to obtain F1 generation heterozygous mice. Mutated F1 mice were crossed and got F2 generation homozygous mice. Genotype and phenotype of the knockout mice were identified by sequencing, flow cytometry and xenograft model. Results Rag2?sgRNA and IL2rg?sgRNA recombination plasmids were constructed and transcribed into RNA. After microinjection and mat? ing, F0 founders were born and F2 homozygous mice were obtained. The results of sequencing showed that there were two types of genotype in IL2rg gene, 10 bp or 11 bp deletion;however, there was only one genotype in Rag2 gene, which was 8 bp deletion. Compared with wild?type BALB/c mice, the number of CD3 +, B220 + and NKp46 + cells in peripheral blood of the knockout mice was reduced significantly. After inoculation of human breast cancer cell line SKBR?2HL cells, tumor size in the xenograft mouse model was increased gradually along with time extension. Conclusion CRISPR/Cas9 is an efficient way to mutate Rag2 and IL2rg gene in mice in vivo, leading to aberrant T cells, B cells and NK cells.
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According to relevant national laws and regulations, practitioner training was included into laboratory animal science teaching reform.By adjusting the training content and teaching method and use of animal models of typical human diseases, the transformation of training mode was realized and improved.By the assessment of basic theory in combination with practical operation, the thinking ability and hands-on skill of the practitioners are much improved. Through classroom instruction, experimental teaching, quality assessment and tracking survey, the evaluating process of the training quality of training teaching is performed.Therefore, the teaching reform of the laboratory animal science based on the training of practitioners is established.
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Objective To establish a patient-derived gastric cancer xenograft( PDX) model in nude mice and to in-vestigate the application of near infrared fluorescent ( NIRF) dye IR-783 in in vivo imaging of gastric cancer xenograft mod-els.Methods Fresh human gastric cancer tissue was taken and transplanted into the subrenal capsule of nude mice to es-tablish the xenograft model.When the transplanted tumors grew,took part of the tumor tissue to do HE staining and compare the structural characteristics with the primary tumor.Another portion of the tumor was xenografted into nude mice subcutane-ously.Twenty days later,the tumor-bearing mice were injected intraperitoneally with IR-783 dye (10μM) in a dose of 100 mg/20 g.The intensity of the tumor image was monitored by optical NIRF imaging.The correction between tumor volume and fluorescence intensity was analyzed.Finally,the expression of OATP1B3 and HIF1αin the xenografted tumor tissue was detected by immunohistochemistry.Results We successfully established three patient-derived xenograft ( PDH) models of human gastric cancer.The transplanted tumor tissues maintained the histological characteristics of the primary tumor well.NIRF signal can be detec ted in subrenal capsule of the xenografted nude mice.The correlation between tumor size and fluorescence intensity in the PDX models reached higher than 98%.Strong positive expressions of HIF1αand OATP1B3 in the tumor tissues were detected.Conclusions NIRF dye IR-783 can be specifically accumulated at the tumor site,therefore, can be used to detect PDX in vivo early.The tumor targeting property may be related to the expression of OATP1B3 and HIF1α.
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Prostate cancer is one of the most common malignant tumors in men and related studies have achieved great breakthrough in recent years.But because of the lack of effective in vivo animal models, the process to translate basic research into clinical application has been severely hampered.Patient derived prostate tumor xenograft ( PDPTX) model is an ideal animal model in which freshly isolated tumor tissues from patients were inoculated into immunodeficient mice.This model can duplicate the heterogeneity of primary tumor in a better way and keep the tumor complexity at molecular, genetic and pathological levels.Particularly, the PDPTX model, in which the isolated tumor tissue is inoculated under the renal capsule, is even better, because it solves the clrawbacks of traditional subcutaneous inoculation model.In traditional mod-els, the success rate is low, it’s not easy for lower grade tumor to form xenograft, and it’s not easy to reconstruct metasta-sis, etc.PDPTX provides a more ideal in vivo model for prostate cancer studies.It has irreplaceable advantages, especially in target therapy, new drug screening and individualized tumor treatment.
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<p><b>OBJECTIVE</b>To investigate the associations between vegetables and fruit intake and quality of life in breast cancer patients.</p><p><b>METHODS</b>A total of 3 344 community breast cancer patients were selected through cluster sampling method between April and July 2013, in Shanghai, China. Data were collected using a questionnaire, which included socio-demographic situation, cancer survival and health behaviors(i.e. vegetables or fruit intake, exercise), European Organization for Research and Treatment (EORTC) QLQ-C30 Simplified Chinese version(3rd edition) and Functional Assessment of Cancer Therapy scale (FACT-G) Simplified Chinese version(4th edition) were used to evaluate the quality of life. Crude quality of life scores were compared between groups. Multiple linear models were used to calculate and compare adjusted means of quality of life between groups, controlling relevant factors.</p><p><b>RESULTS</b>After adjusting relevant factors, breast cancer patients who ate more than 250 g vegetables reported higher EORTC physical functioning scores, cognitive functioning scores, emotional functioning scores, global health scores than patients who ate equal or less than 250 g vegetables(respectively (80.79 ± 0.85) vs (79.34 ± 0.82), (80.07 ± 1.03) vs (77.84 ± 0.99), (84.17 ± 0.95) vs (82.76 ± 0.92), (65.75 ± 1.50) vs (62.92 ± 1.45)), t values respectively were 2.76, 3.54, 2.40, 3.17, all P values were <0.05; and breast cancer patients who ate more than 250 g vegetables reported higher FACT-G social well-being scores, function well-being scores, FACT-G total scores than patients who ate equal or less than 250 g vegetables (respectively (17.92 ± 0.40) vs (17.31 ± 0.39), (14.86 ± 0.42) vs (14.34 ± 0.40), (74.78 ± 1.01) vs (73.05 ± 0.97)), t values respectively were 2.49, 2.05, 2.90, all P values were <0.05. After adjusting relevant factors, breast cancer patients who ate fruit everyday reported higher EORTC physical functioning scores, role functioning scores, cognitive functioning scores, emotional functioning scores, social functioning scores, global health scores than patients who didn't eat fruit everyday (respectively (80.40 ± 0.82) vs (79.22 ± 0.87), (89.81 ± 1.00) vs (88.06 ± 1.05), (79.78 ± 0.99) vs (77.11 ± 1.04), (84.43 ± 0.92) vs (81.56 ± 0.97), (77.95 ± 1.25) vs (75.56 ± 1.31), (65.48 ± 1.44) vs (61.74 ± 1.51)), t values respectively were 2.15, 2.64, 4.07, 4.71, 2.89, 4.02, all P values were <0.05; and breast cancer patients who ate fruit everyday reported higher FACT-G physical well-being scores, social well-being scores, emotional well-being scores, functional well-being scores, FACT-G total scores than patients who didn't eat fruit everyday(respectively (23.35 ± 0.26) vs (22.85 ± 0.28), (17.91 ± 0.39) vs (16.98 ± 0.41), (18.59 ± 0.22) vs (18.18 ± 0.23), (14.79 ± 0.40) vs (14.17 ± 0.42), (74.71 ± 0.97) vs (72.17 ± 1.02)), t values respectively were 2.92, 3.65, 2.91, 2.35, 4.05 , all P values were <0.05.</p><p><b>CONCLUSION</b>There are active associations between vegetables / fruit intake and quality of life in breast cancer patients. Proper diet may help improve quality of life in breast cancer patients.</p>
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Female , Humans , Breast Neoplasms , China , Diet , Feeding Behavior , Fruit , Quality of Life , Surveys and Questionnaires , VegetablesABSTRACT
Objective To evaluate the effect of near infrared heptamethine cyanine dye IR-783-mediated specific tumor imaging in spontaneous tumor of dogs .Methods IR-783 was intraperitoneally injected to nude mice models of transplanted tumor in a dose of 5μmol/kg.The metabolic time course of IR-783 was detected by in vivo imager .Based on the results of above observation , IR-783 was injected to dogs with spontaneous tumor in a dose of 1.5μmol/kg.The site of intravenous injection was the hind leg .Tumor and peri-tumoral tissues were removed at 5 days after IR-783-injection for fluorescence imaging , pathology and frozen section fluorescence examinations .Results After i.p.IR-783 injection to nude mice models of transplanted tumor , the transplanted tumor tissues of nude mice had stronger specific fluorescence than normal tissues by imaging at 8 days after injection .After i.v.IR-783 injection to four dogs with spontaneous tumor , the fluorescence signal in the tumor tissues was stronger than that in the normal tissues at 5 days after injection .Conclusions Near infrared fluorescent dye IR-783 could be specifically taken up by tumor tissues , and can be used for specific diagnosis of tumor.It has an important clinical application prospect .
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<p><b>OBJECTIVE</b>To investigate the associations between physical exercise and quality of life in breast cancer patients.</p><p><b>METHODS</b>A cross-sectional study was conducted among 3 344 community breast cancer patients between April and July 2013 in Shanghai, China. Data were collected using a questionnaire, including socio-demographic situation, cancer survival and health behaviors, and scores of EORTC QLQ-C30 Simplified Chinese version and FACT-G Simplified Chinese version.</p><p><b>RESULTS</b>Among a total of 3 344 breast cancer patients, the patients doing exercise reported significantly higher EORTC physical functioning scores, role functioning scores, emotional functioning scores, global health scores, and FACT-G physical well-being scores, social well-being scores, emotional well-being scores, functional well-being scores, and FACT-G total scores than the patients who didn't take exercise (P < 0.05, P(Adjusted)<0.05) . Breast cancer patients who did exercise more than or equal to 5 times/week reported significantly higher EORTC role functioning scores, cognitive functioning scores, emotional functioning scores, global health scores and FACT-G physical well-being scores, functional well-being scores, and FACT-G total scores than patients who did exercise less than 5 times/week (P < 0.05, P(Adjusted)<0.05 ).</p><p><b>CONCLUSIONS</b>There are active associations between physical exercise and quality of life in breast cancer patients. Engagement in physical exercise is beneficial to breast cancer patients with long-term survival.</p>
Subject(s)
Humans , Asian People , Breast Neoplasms , Epidemiology , China , Epidemiology , Cross-Sectional Studies , Exercise , Quality of Life , Surveys and QuestionnairesABSTRACT
Objective To construct a recombinant bacillus Calmette-Guérin(BCG) vaccines based on different tandem repeats of MUC1 and GM-CSF, rBCG-MVNTR1/4/8-CSF, and to observe the ability of three recombinant BCG vaccines in the inhibition of breast cancer. Methods After MUC1 variable-number tandem repeats (MVNTR1/4/8) were cloned in a stepwise manner, the E. coli-Mycobacteria shuttle expression vector pDE22-MVNTR1/4/8-CSF were constructed by fusing MVNTR1/4/8 and GM-CSF, and then used to transform competent BCG by electroporation after identification by restriction endonuclease digestion analysis and DNA sequencing. A novel breast cancer vaccines, rBCG-MVNTR1/4/8-CSF was constructed. The expression of fused MVNTR1/4/8-CSF protiens was analyzed by SDS-PAGE and Western blot. The ability of rBCG vaccines inhibiting the growth of breast cancer was observed in hu-PBL-SCID mice. The specific T cell responses in mice were assessed by immunohistochemistry. Results The expression of recombinant MVNTR1/4/8-CSF fusion proteins were detected by SDS-PAGE and Western Blot in rBCG-MVNTR1/4/8-CSF vaccines, respectively. Tumor incidence in mice prophylactic immunized with rBCG-MVNTR4-CSF (37.5%) or rBCG-MVNTR8-CSF (25%) significantly decreased compared with PBS and BCG-pDE22 control ( 100% ) at 42 days after tumor implantation ( P ＜ 0. 05 ). MCF-7 tumor growth inhibition in rBCG- MVNTR4/8-CSF-immunized mice was more significant than that in controls ( P ＜0. 01 ).The inhibition effect of three rBCG vaccines on breast rumor growth appeared to rise with increase of numbers of the tandem repeats of MUC1. Survival rate was 75% of mice in the rBCG-MVNTR4-CSF-treated group and 87. 5% of mice in the rBCG-MVNTR8-CSF-treated groups at 70 days after tumor implantation; however,survival rate was only 12. 5% in control group( P ＜0. 05). The CD4-positive and CD8-positive lymphocytes were detected only in rBCG-MVNTR4/8-CSF-immunized mice. Conclusions rBCG-MVNTR4/8-CSF immunization inhibits breast cancer growth in mice.
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Objective To purify native and recombinant heparin-binding hemagglutinin(HBHA)protein,and investigate the activity of HBHA polyclonal antibody against aggregation of Bacillus CalmetteGuerin(BCG)induced by HBHA.Methods After growing BCG to the stationary phase in the 7H9 liquid medium,the native HBHA protein(nHBHA)was obtained by CL-6B column chromatography.At the same time,the HBHA gene fragment was cloned and expressed by transforming Escherichia coli BL-21.Then the polyclonal antibody against rHBHA was prepared by immunizing rabbit.Different comcentration of the HBHA protein was added to the BCG liquid medium,and the aggregation of the BCG was observed.Then,add the HBHA protein that incubated with anti-HBHA antibodies to the BCG culture medium and observe the aggregation of BCG.Results The purity of native HBHA was 99% and the concentration was 1.016 mg/ml.The expressed product contained 36% of total somtic protein.After purified,the purity of the recombinant HBHA protein was 97.1% and the concentration was 10.98 mg/ml.Both the rHBHA and nHBHA could induce the aggregation of BCG.When then concentration of nHBHA is 0.2μg/ml,BCG could be induced to aggregate,while the rHBHA concentration is 2μg/ml could induce the aggregation.Both aggregations could be suppressed by the polyclonal antibody against rHBHA.Conclusions The native and recombinant HBHA are successfully obtained.It is proved that the rHBHA could induce the aggregation of BCG similar as nHBHA,and polyclonal antibody against rHBHA could also suppress the activity of nHBHA.It suggested that rHBHA could be further used in clinical diagnosis and vaccination.
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Objective To purify Micrococcus luteus Rpf and Rpf domain fusion protein, and to in-vestigate its effects on growth of Mycobacterium tuberculosis. Methods The recombinant plasmids pPro-EXHT-Rpf and pPro-EXHT-Rpf domain were expressed in E. Coli DHSa and then purified under denaturing condition via Ni-NTA purification system and confirmed by Western blot. The biochemical property of the M. Luteus Rpf and Rpf domain was analyzed by stimulating the resuscitation of M. Tuberculosis H37Ra which were in non-culturable' condition. Results The Rpf and Rpf domain products achieved 95% and 93% pure respectively, and the molecular weight was 30 x 103 and 12 x 103, the yield of purification was about 471 mg/L and 337 mg/L of the culture. The M. Luteus Rpf and Rpf domain from the E. Coli showed activity of stimulating the resuscitation of M. Luteus and M. Tuberculosis H37Ra in non-cuhurable' condition which could be inhibited by monoclonal antibodies of M. Luteus Rpf domain remarkably. Conclusion It was dem-onstrated that the purification of Rpf and Rpf domain have high biological activity for further functional, pharmacological and clinical investigations, and M. Luteus Rpf domain protein is fully active as M. Lateus full-length Rpf.