Human LINE1 endonuclease domain as a putative target of SARS-associated autoantibodies involved in the pathogenesis of severe acute respiratory syndrome / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Severe acute respiratory syndrome (SARS) is a disease with a mortality of 9.56%. Although SARS is etiologically linked to a new coronavirus (SARS-CoV) and functional cell receptor has been identified, the pathogenesis of the virus infection is largely unclear.</p><p><b>METHODS</b>The clinical specimens were processed and analyzed using an indirect enzyme-linked immunosorbent assay (ELISA) in-house. Further investigations of target antigen included reviews of phage display technique, rapid amplification of cDNA ends (RACE) technique, protein expression and purification, Western blotting validation, serological and immunohistochemical staining in postmortem tissue.</p><p><b>RESULTS</b>A type of medium or low titer anti-lung tissue antibodies were found in the sera of SARS patients at the early stage of the disease. Human long interspersed nuclear element 1 (LINE1) gene endonuclease (EN) domain protein was one of the target autoantigens and it was aberrantly expressed in the lung tissue of SARS patients. Anti-EN antibody was positive in the sera of 40.9% of SARS patients.</p><p><b>CONCLUSIONS</b>Human LINE1 endonuclease domain was identified as a putative target of SARS-associated autoantibodies, which were presented in the serum of SARS patients and may be involved in the pathogenesis of SARS.</p>

Screening of the genes of hepatitis B virus e antigen interacting proteins / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To screen and clone the genes in hepatocytes which encode protein that can interact with hepatitis B e antigen(HBeAg) by yeast-two hybridization.</p><p><b>METHODS</b>Recombined HBeAg bait plasmid (pGBKT7-eAg) was transformed into yeast AH l09, followed by mating with yeast Yl87 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-Ade-His) which contains X-a-gal for selecting positive blue clones. Then positive clones were selected and plasmids were prepared and sequenced. Finally, bioinformatics analysis was performed.</p><p><b>RESULTS</b>Totally 245 positive colonies were selected and 101 colonies were sequenced. Through sequences alignment, 6 novel genes and 35 recorded genes were screened.</p><p><b>CONCLUSION</b>Genes of HBeAg interacting proteins have been cloned successfully, which brings some new clues for further studies on the biological functions of HBeAg and the related proteins.</p>

Detection of autoimmune parameter of SARS patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore whether autoimmune phenomena exist in SARS patients, and to seek for unusual autoimmune antibodies in SARS patients.</p><p><b>METHODS</b>Autoantibodies against cell nuclei (ANA), autoantibodies against smooth muscles (SMA), autoantibodies against parietal cells (PCA), autoantibodies against heart cells (HRA) were detected by using immunofluorescence, and autoantibodies against live-kidney microsomes (LKM) and anti-M2 antibodies were detected by ELISA in sera taken from 27 SARS patients and 18 healthy controls. Immunofluorescence was used to localize the targets antigens in slides with biochips of lung (monkey) of SARS associated antibodies.</p><p><b>RESULTS</b>ANA, AMA, LKM and SMA were found positive in 3, 1, 1, and 1 of the 27 SARS sera. In 18 healthy control sera, one ANA and one AMA were positive. Statistical analysis showed that there were no difference between two groups in every item detected. Twenty-six of 27 SARS patients and 5 of 18 healthy controls had strongly stained columnar epithelia of the bronchiole, especially the lumen border of the epithelia?the difference between two groups was significant.</p><p><b>CONCLUSION</b>No antibodies against organs but lung were found in SARS patients. There are auto antibodies against lung tissues in sera of SARS patients.</p>

Construction and expression of DNA-binding domain plasmid with hepatitis B virus e antigen in yeast double hybrid system / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Using hepatitis B virus e antigen (HBeAg) gene to construct the DNA-binding domain vector, which can express HBeAg in yeast cell, and can be used in yeast double hybrid as "bait plasmid" to look for the gene from the cDNA library, which expresses the protein that can interact with HBeAg.</p><p><b>METHODS</b>PCR was performed to amplify the HBeAg gene from a sera of hepatitis B patient. The product of the amplification was inserted into T-vector and was verified by sequencing. Then it was inserted into the "bait" plasmid pGBKT7 after the digestion with the restricted endonuclease of EcoR I and Sal I. The plasmid was transformed into the yeast cell. PCR was used to verify whether the plasmid was transformed into yeast. The HBeAg protein expressed in the cell was confirmed by Western blot. Using nutrition selection assay to verify the constructed plasmid alone could not activate the reporter gene in the yeast cell.</p><p><b>RESULTS</b>Sequenced and digested by two endonucleases, the recombined vectors pGBKT7-eAg produced anticipated fragment. PCR verified that there was HBeAg fragment in the yeast. Having assayed by Western blotting, it was shown that the yeast cell transformed with pGBKT7-eAg vector had positive signal which could not be seen in the control. Tested by the nutrition selection assay, the recombined vectors pGBKT7-eAg could not activate LacZ reporter gene in the yeast.</p><p><b>CONCLUSION</b>DNA-binding domain plasmid was successfully constructed and could express HBeAg proteins in the yeast cell but could not activate transcription of LacZ reporter gene alone. The recombined plasmid can be used in yeast double hybrid.</p>

Cloning and expressing the E2 subunit of pyruvate dehydrogenase complex / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To construct the expression vector of the pyruvate dehydrogenase complex E2 subunit gene (PDC-E2).</p><p><b>METHODS</b>The PDC-E2 gene was amplified from human lymphocytes with RT-PCR, and was cloned into pExSecI vector to induce the PDC-E2 expression. The products were identified with western blot and ELISA.</p><p><b>RESULTS</b>The expression vector pExSecI/PDC-E2 was successfully constructed. The products could be identified by the specific self-antibodies in the sera from the primary biliary cirrhosis patients.</p><p><b>CONCLUSION</b>High efficient expression vector of PDC-E2 lays the foundation for serum assay of primary biliary cirrhosis patients with prokaryotic expressing PDC-E2.</p>