Biomedical subjects

Zhi-Yong He

Publications and source records attributed to Zhi-Yong He.

8 recordsLinked to original sources

PubMed2003

None of the integrins known to be present on the mouse egg or to be ADAM receptors are essential for sperm-egg binding and fusion.

Antibody inhibition and alpha6beta1 ligand binding experiments indicate that the egg integrin alpha6beta1 functions as a receptor for sperm during gamete fusion; yet, eggs null for the alpha6 integrin exhibit normal fertilization. Alternative integrins may be involved in sperm-egg binding and fusion and could compensate for the absence of alpha6beta1. Various beta1 integrins and alphav integrins are present on mouse eggs. Some of these integrins are also reported to be receptors for ADAMs, which are expressed on sperm. Using alpha3 integrin null eggs, we found that the alpha3beta1 integrin was not essential for sperm-egg binding and fusion. Oocyte-specific, beta1 integrin conditional knockout mice allowed us to obtain mature eggs lacking all beta1 integrins. We found that the beta1 integrin null eggs were fully functional in fertilization both in vivo and in vitro. Furthermore, neither anti-mouse beta3 integrin function-blocking monoclonal antibody (mAb) nor alphav integrin function-blocking mAb inhibited sperm binding to or fusion with beta1 integrin null eggs. Thus, function of beta3 or alphav integrins does not seem to be involved in compensating for the absence of beta1 integrins. These results indicate that none of the integrins known to be present on mouse eggs or to be ADAM receptors are essential for sperm-egg binding/fusion, and thus, egg integrins may not play the role in gamete fusion previously attributed to them.

ADAM Proteins↗

PubMed2001

A Candidate Oral Vaccine to Helicobacter pylori Fusion Protein of HspA and CtxB.

Heat-shock protein A subunit(HspA), an effective immunogen may stimulate the immunoresponse in human body against challenge of H.pylori. The B subunit of cholera toxin (CtxB) has been proved to be a potent mucosal immunogen, actas an adjuvant for vaccine targeted for delivery to the mucosal-associated lymphoid tissue. A recombinant plasmid expressing bivalent antigen of HspA and CtxB subunit was constructed as follows. hspA and ctxB gene was amplified by PCR. The DNA products of hspA and ctxB were inserted in the prokaryotic expression vector pET-22b( ), respectively, and then the resulted recombinant plasmid expressinga fusion protein named HCT was transformed into the E.coli strain BL-21(DE3). hct gene was measured to be 708 base pairs long, and the fusion protein encoding a polypeptide of 236 amino acid residues, corresponded to a calculated molecular masses of 30 kD. Western blot analysis of the recombinant protein HCT confirmed that it could be specifically recognized by the serum of H.pylori-infected patients. HspA and HCT labelled (125)I were orally administered into the stomach of mice, respectively, and the radioactivity of (125)I in serum of each mouse was assayed at intervals 15 min, 30 min, 60 min, 90 min and 120 min. The result indicated that there were high radioactivity counts in the groups of HCT than that of HspA(P 0.001). This result suggests that the CtxB may enhance the volume of HspA absorbed from the intestine of mice, therefore the recombinant fusion protein HCT may be an effective oral vaccine for prevention and treatment against the infection of H.pylori.

Journal Article↗

PubMed2000

Cloning and Expression of Tetanus Toxin Fragment C in E.coli.

The fragment C of tetanus toxin was amplified from Clostridium tetani DNA by PCR. This fragment was cloned into expression vector pET-28a(+),under the control of the T7 promoter. Expression of this plasmid in E.coli resulted in the production of a protein consisting of 6xHis of the vector fused to the N-terminal 451 amino acids of tetanus toxin. After induction with 1 mmol/L IPTG, TTC was expressed in E.coli BL21(DE3). The protein product accounted for 8.2% of the bacteria total protein in soluble form, SDS-PAGE and Western blot analysis of TTC recombinant protein confirmed this result. The expression products were also purified by Ni(2+)-IDA-Sephrose 6B column. Immunization of mice with rTTC resulted in the production of antibodies that were able to protect mice against a challenge with tetanus toxin furthermore, rTTC in vivo appeared to be able to undergo retrograde axonal transport.

Journal Article↗

PubMed2000

Cloning, Expression and Tumor Suppression of Human Endostatin.

Human endostatin cDNA was cloned from total RNA of normal Chinese liver cell line L02 by RT-PCR. Endostatin DNA sequence encoded 184 amino acid residues. Five base pairs and 3 amino acid residues are different from that reported, it may be due to interspecies difference. The endostatin cDNA was inserted into the pET-28a(+) containing T7 promoter. The recombinant plasmid was transformed the E.coli BL21(DE3). Recombinant human endostatin was highly expressed as inclusion body when the expression strain BL-ENDO was induced with 1 mmol/L IPTG. Result of SDS-PAGE analysis revealed that recombinant human endostatin was accounted for up to 25% of soluble protein in E.coli. Purified and refolded recombinant human endostatin was active in inhibiting tumor growth and metastasis.

Journal Article↗

PubMed2000

A Structure-function Analysis of Human GDNF.

The glial cell line-derived neurotrophic factor(GDNF) plays a very important role in the regeneration of the nervous system. Based on the results of the X-ray structure analysis of rat GDNF, human GDNF gene was modified with deletion and insertion mutagenesis by using PCR methods. The various mutants were all highly expressed in E.coli. The recombinant proteins were purified and their survival-promoting activities were determined by using cultures of the spinal cord neurons of embryonic mouse. The results showed that the "cystine knot motif" was critical for the maintenance of GDNF structure the alpha-helix, finger 1 and finger 2 region were critical for GDNF neurotrophic activity and the N-terminus of human GDNF was not essential for its biological functions.

Journal Article↗

PubMed2000

High Expression of Human Persephin in Insect Cells.

The human persephin (PSP) was expressed in Tn-5B1-4 insect cells using the Bac to Bac baculovirus expression system. The expressed product amounted for 20% of total cellular soluble proteins. The expressed product was purified by Ni(2+) affinity chromatography and the activity assays showed that it could significantly prolong the survival of spinal cord neurons.

Journal Article↗

PubMed2000

Cloning of the CtxB Gene of Vibrio cholerae and Its Expression in E.coli.

The CtxB gene encoding cholerae toxin subunit B was amplified from Vibrio cholerae genomic DNA by PCR. The result of sequencing indicated that CtxB gene encodes 124 amino acid residues. The sequence of CtxB gene was almost the same as that of reported except for the codon of Thr 62. The expression plasmid pGEX-CTXB was constructed by inserting CtxB gene into plasmid pGEX-4T-2, containing gst gene, immediately downstream of the T7 promoter. The expressed plasmid was introduced into E.coli BL21(DE3) cells and expression strain CTXB/BL21 was selected. SDS-PAGE analysis revealed that the GST-CTXB fusion protein was highly expression and accumulated up to 36% of bacterial soluble proteins after the induction by IPTG. A fusion protein of 40 kD was expressed as inclusion body. The fusion protein was refolded and purified. The purified fusion protein was cut by thrombin to obtain the purified CTXB protein.

Journal Article↗

PubMed1999

Cloning and Expression of a Novel Mutated Osteoprogerin/Osteoclastogenesis Inhibitory Factor Gene.

Total RNA was isolated from normal Chinese human liver cell line L02. A mutated osteoprotegerin/osteoclastogenesis Inhibitory Factor(OPG/OCIF) cDNA was amplified by RT-PCR using the total RNA as template and was inserted into pBS-sk plasmid. Sequence analysis showed that the OPG/OCIF cDNA from L02 cells was a mutated OPG/OCIF gene which had a nonsense mutation(Ochre) at the codon of Gln(394). The OPG/OCIF isoform was 8 amino acid residues less at the C terminal than the OPG/OCIF reported. The 3'fragment of OPG/OCIF gene from genomic DNA of cell line 293(ATCC CRL 1573) was also cloned. Sequence analysis indicated that the sequence of genomic DNA was the same as that of cDNA. The mutated OPG/OCIF gene was inserted into yeast expression plasmid pPIC3.5K, and the recombinant plasmid was used to transform Pichia pastoris GS115. SDS-PAGE analysis revealed that the human OPG/OCIF isoform was highly expressed and accumulated up to over 30% of soluble protein of yeast after the induction by methanol for 3 to 5 days. The longer the transformant was induced, the higher the ratio of glycosylated protein was.

Journal Article↗