Human Bsp Protein Cdna Cloning, Localization And Expression

BSP ( boyine seminal plasma) protein is the cardinal protein in bovine sperm, which can combine with choline or phosphycholine and then adhere to the surface of sperm to promote the sperm capacitation induced by heparin and high density lipoproteins ( HDL ). Moreover, BSP protein can combine with heterogeneous collagen, fibrinogen, heparin, calmoduline, IGF - II and apoA - I . BSP can also inhibit the migration of cholesterol on the sperm membrane. All these indicate that BSP protein plays an important role in the lipid metabolism of sperm membrane and the modulation of sperm capacitation. At present, homogeneous proteins of BSP proteins found in the sperm plasma of horse and pig have the binding - properties similar to BSP protein, which indicates that BSP protein and its homogeneous protein exist commonly in mammals and show their physiological functions. However, in human congenital system proteins associated with BSP in function have not been reported up to date. To find BSP proteins and its related proteins important roles in fertilization and the development of the zygote, we clone the full - length cDNA which is related to BSP genes and named HBRP( Human BSP - Related Proteins). We can not find the same protein in Genbank through the BLAST procedure from NCBI. We located HBRP gene on the chromosome and examined the expression of HBRP gene in human tissues. We also expressed HBRP in E. Coli. We have obtained amount of expression proteins through the gene recombinant technique and detected its effect on the activity of PKC, which is crucial to further study.Materials and methods1. 3'-RACE and 5'-RACE PCR reactionTwo specific 3'- RACE primers are designed by the reference to the ESTcDNA sequence ( AA429491) from GenBank date House of human testis tissue. Using Human Testis Marathon - Ready cDNA as template, the 3'- RACE PCR reaction is performed by using the Marathon?cDNA Amplification Kit and Advantage cDNA Polymerase. On the basis of above work, 5'- RACE PCR reaction primer is designed. 5'- RACE PCR reaction is followed and analyzed in 1% agarose/EtBr gel.2. Cloning and analyzing of the full -length cDNA sequenceThe fragments consistent with expected length from the product of RACE -PCR reaction are separated, purified and recovered, after which the fragments are cloned into the vector of pMD - 1ST to transform the competent cells. The positive clone is selected by colony PCR and DNA sequence is analyzed and determined by the M13 primer. Then the correct sequence is determined by the computer processing. The full - length cDNA of HBRP is amplified by PCR reaction and sequenced after cloning.3. Computer analysis of the cDNA and its encoding proteinThe amino acid sequence encoded by full - length cDNA is deduced by u-sing the BLAST procedure from NCBI. The homogeneous comparison and analysis are performed in the GenBank and protein Date House, while the functional residual sequence of the protein is analyzed in the molecular Biological net, Ht-tp://www. expasy. ch/cgi - bin/scanpaosite4 . Location of the HBRP gene in the chromosomeAccording to Stanford G3 RH panel, a pair of primers are designed in the 3' - UTR region of the gene which is to be localized and human genome DNA is amplified in advance whose products are examined by electrophoresis. Then G3 panel is amplified and this process is repeated twice. The PCR result is sent to Stanford Human Genome Center (SHGC) http://www - shgc. Stanford, edu/ RH/rhserverformnew. html, obtaining the nearest label number of SHGC to the gene which is to be localized, eventually, finding the corresponding chromosome band through the RH graphics ahd genome hereditary graphics.5. RT-PCRThe total RNA of normal tissues is extracted by using the method of TrizolReagent. The first chain of cDNA is synthesized through reverse transcriptase. The primers are designed in sites of the number 2 and 3 exon of HBRP gene. The product of RT - PCR by using human B - actin as primer is looked on as the positive control. The result i...