BACKGROUND: The human leucocyte antigen (HLA) allele are now understood to be alternative DNA sequences at the same physical gene locus, which may or may not result in different phenotypic traits. The generation of a new allele is induced by various factors, but, whether the mutation would be exist after removing the causative factors need further investigation. OBJECTIVE: To confirm the new allele by DNA sequencing technique, in addition, to analyze the heritage of a HLA allele A~*9217 (No. of WHO registration: WHS10004629)DESIGN, TIME AND SETTING: The open experiment with DNA as observation object. The initial detection with polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) was performed at HLA Laboratory of Henan Provincial Red Cross Blood Center in November 2007, and the sequencing was performed at HLA Laboratory of DYNAL Biological Technology (Beijing) Co., Ltd. in February, 2008. MATERIALS: The proband (sample ID: 371xxxxx ) and other 8 family member was investigated, blood sample was collected at the HLA Laboratory.METHODS: Nine family members of A~*9217 carrier were typing for HLA- A, B, DRB1 using PCR-SSO and SBT methods for low-media and high resolution, and 3 red blood cell blood groups systems: ABO, MN and Rh was tested to assist analysis .MAIN OUTCOME MEASURES: Sequence alignment of HLA-A exon 3 in A~*9217 carriers.RESULTS: According to the results of the blood groups phonotype of ABO, MN, Rh systems and HLA, the new allele A~*9217 of the proband was paternal origin. However, the sequence of this allele is A*020301, which had 3 base difference in exon 3, the new allele appears 3 base change at 391 T>G, 414 C>G, 418 T>G, and this new sequence was found in proband's two children.CONCLUSION: The new allele A~*9217 is generated from proband's father mutation, which can pass it to his children. However, the further heritage of A~*9217 allele need exploring.

In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.
Animals ; Animals, Genetically Modified ; genetics ; Cloning, Organism ; veterinary ; Gene Knockout Techniques ; Gene Targeting ; methods ; Genes, Reporter ; Genetic Engineering ; Genetic Vectors ; genetics ; Goats ; genetics ; Integrases ; chemistry ; metabolism ; Recombination, Genetic ; Transgenes ; genetics

In the application of therapeutic antibodies, large molecular weight of antibodies is always a problem that prevents them from penetrating into tissues or binding to antigenic determinants. To overcome this problem, we investigated the function of the heavy chain variable domain of a monoclonal anti-VEGF human IgM antibody derived from the Five-Feature Translocus Mice. We cloned the cDNA of the heavy chain variable domain, which was then inserted into pET28a vector and expressed in Escherichia coli. After purification and renaturation of the denatured recombinant protein, we obtained a 16 kDa antibody fragment, which is named as rhVVH. By immunoassaying its VEGF-binding capability in vitro, we proved that rhVVH retains this activity as the complete IgM. Importantly, rhVVH is shown to inhibit the HUVEC cell proliferation in a concentration-dependent manner. Our results indicate that the single heavy chain variable domain might inherit part of the biological function of the complete IgM antibody, which provided a valuable potential in further research on antibody miniaturisation.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Heavy Chains ; biosynthesis ; genetics ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; Mice ; Molecular Sequence Data ; Recombinant Proteins ; biosynthesis ; genetics ; Single-Chain Antibodies ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Objective To evaluate the bacteriostatic effect of recombinant human lactoferrin(rhLF) on Helicobacter(H .) py‐lori and its influence on CagA ,Ure and gastric mucosal IL‐8 .Methods The minimum inhibitory concentration(MIC)and the influ‐ence of different drug concentrations on the proliferation of H .pylori were detected .The effects of rhLF on the mRNA and protein expressions of CagA and Ure in H .pylori were detected by RT‐PCR and Western blot ,respectively .The animal study :Balb/c mice were adopted and assigned randomly into four groups ,including the standard triple+rhLF(group A) ,rhLF(group B) ,standard tri‐ple(group C) and normal saline(group D) .The histopathological HE staining was used to observe the gastric inflammation and ELISA was used to detect the IL‐8 level of gastric tissue in each group .Results MIC was 0 .5 mg/mL ,moreover rhLF inhibited the bacterial growth and proliferation with a concentration‐dependent manner .rhLF could reduce the expression of H .pylori major viru‐lence factor CagA ,mRNA and protein of Ure .Comparing the group A with the group B ,C and D ,the gastric mucosal inflammation score and the IL‐8 levels of gastric tissue homogenates had statistically significant differences(P<0 .05) .Conclusion rhLF inhibits the growth and proliferation of H .pylori ,moreover inhibit the expression of major virulence factor CagA in H .pylori ,mRNA and protein of Ure in different degrees ,weakens its pathogenicity ,meanwhile reduces the IL‐8 level in mice gastric mucosa ,and allevi‐ates H .pylori related gastric mucosal inflammatory response .

Objective:To compare the values of medical image technologies in evaluating the tansperineal laser ablation (TPLA) in canine prostate.Methods:TPLA (3 W/600 J and 3 W/1 200 J) were operated in the prostate of six adult male beagles guided by transrectal ultrasound (TRUS). TRUS, transrectal contrast-enhanced ultrasound (TR-CEUS) and multiparameter magnetic resonance imaging (mpMRI) were used to evaluate the ablation on the day of TPLA, one week and one month after TPLA. The animals were sacrificed for pathology to calculate the volume of the ablation. SPSS 22.0 software was used for statistical analysis.Results:TRUS could be used to guide and observe the puncture and ablation process during TPLA. TR-CEUS and contrast enhanced MRI showed good consistency in the volume of ablation ( P>0.05). One month after TPLA, the ablation volume were (1.69±0.51)ml vs (1.73±0.36)ml vs (1.52±0.41)ml (3 W/600 J) and (2.23±0.54)ml vs (2.34±0.29)ml vs (2.19±0.34)ml (3 W/1 200 J) measured by the two medical image technologies and pathology, with good consistency ( P>0.05). Conclusions:TRUS can be used to guide and observe the puncture and ablation process during TPLA. TR-CEUS and mpMRI can be used for postoperative evaluation and follow-up of TPLA. The former has advantages of real-time and low price, which can be promoted and applied in clinical practice.